Amyloid binding agents

ABSTRACT

There are provided compounds and methods for the detection of amyloids and treatment of diseases related to amyloids including Alzheimer&#39;s disease and other related amyloid-based neurodegenerative diseases.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 61/285,470,filed Dec. 10, 2009 (Attorney Docket No. 021935-003900US), the entirecontent of which is incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No.1E21RR025358 awarded by National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

BACKGROUND

Alzheimer's disease (AD) is characterized by a progressive loss ofcognitive function and constitutes the most common and fatalneurodegenerative disorder. Genetic and clinical evidence supports thehypothesis that accumulation of amyloid deposits in the brain plays animportant role in the pathology of the disease. This event is associatedwith perturbations of biological functions in the surrounding tissueleading to neuronal cell death, thus contributing to the diseaseprocess. The deposits are composed primarily of amyloid (Aβ) peptides,typically a 39-43 amino acid sequence that self aggregates into afibrillar β-pleated sheet motif. While the exact three-dimensionalstructure of the aggregated Aβ peptides is not known, a model structurethat sustains the property of aggregation has been proposed. Thiscreates opportunities for in vivo imaging of amyloid deposits that notonly can help evaluate the time course and evolution of the disease butcan also allow the timely monitoring of therapeutic treatments.

Historically, Congo Red (CR) and Thioflavin T (ThT) have provided thestarting point for the visualization of amyloid plaques and are stillcommonly employed in post mortem histological analyses. However, due totheir charge these compounds are thought to be unsuitable for in vivoapplications. To address this issue, several laboratories developedcompounds with non charged, lipophilic (log P=0.1-3.5) and low molecularweight chemical structures (M.W. less than 650) that facilitate crossingof the blood brain barrier. Further functionalization of these compoundswith radio-nuclides led to a new generation of in vivo diagnosticreagents that target plaques and related structures for imaging withpositron emission tomography (PET) and single-photon emission computedtomography (SPECT), as known in the art.

Despite these advances, there is a pressing need for the design anddevelopment of new amyloid-targeting molecules with improved physical,chemical and biological characteristics. Provided herein are methods andcompounds addressing these and other needs in the art.

BRIEF SUMMARY

Herein are provided inter alia compounds and methods for the detectionof amyloids and treatment of diseases related to amyloids includingAlzheimer's disease and other related amyloid-based neurodegenerativediseases.

In a first aspect, compounds that bind amyloid peptides and/or amyloids(e.g., amyloid peptide aggregates) are provided. In some embodiments,the compounds described herein have the structure of Formula (I):

In Formula I, “EDG” is an electron donating group. The term “πCE” is api-conjugation element. “WSG” is a water soluble group.

In another aspect, there is provided a pharmaceutical composition. Thepharmaceutical composition includes a compound described herein and apharmaceutically acceptable excipient.

In another aspect, there is provided a method of detecting an amyloidpeptide and/or an amyloid. The method includes contacting a compound asdescribed herein with an amyloid peptide thereby forming a detectableamyloid complex, and detecting the detectable amyloid complex.

In another aspect, there is provided a method of treating a diseasecharacterized by an accumulation of amyloids (e.g., amyloid deposits) ina subject. The method includes administering to a subject in need oftreatment an effective amount of a compound or pharmaceuticalcomposition as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D depict fluorescence excitation and emission spectra of Cmpds8d and 11 in aqueous PBS solution (solid lines) and in the presence ofAβ peptide (dashed lined). FIG. 1A: fluorescence excitation spectrum ofCmpd 8d. FIG. 1B: emission spectrum of Cmpd 8d. FIG. 1C: fluorescenceexcitation spectrum of Cmpd 11. FIG. 1D: emission spectrum of Cmpd 11.

FIG. 2 depicts the apparent binding constant (K_(d)) of Cmpds 8d and 11to preaggregated Aβ peptide. Legend: Cmpd 8d (diamond); Cmpd 11 (box).

FIG. 3 depicts the inhibition of IgG-Aβ fibril interactions with Cmpd 8d(FIG. 3 top) and Cmpd 11 (FIG. 3 bottom).

FIG. 4 depicts fluorescence excitation spectra (left panels) andemission spectra (right panels) for Cmpds 8a, 8b, 8c, 14 and 19 withaggregated Aβ(1-42) fibril, as described herein.

FIG. 5 depicts fluorescence emission spectra of Cmpds 8a, 8b, 8c, 8d,11, 14 and 19 with and without monomeric Aβ monomer, as describedherein.

FIG. 6 depicts a double reciprocal plot of fluorescence maxima andconcentration of Cmpds 8a, 8b, 8c, 14 and 19, as described herein.Legend: Cmpd 8a (diamond); Cmpd 8b (box); Cmpd 8c (triangle); Cmpd 14(cross); Cmpd 19 (barred cross).

FIGS. 7A-D depict inhibition maxima and 1E₅₀ values for compoundsdescribed herein. FIG. 7A: Cmpd 8a; FIG. 7B: Cmpd 8b; FIG. 7C: Cmpd 8c;FIG. 7D: Cmpd 14.

FIG. 8 depicts the results of cytoxicity studies as described herein.Legend: for each concentration of compound employed in the cytoxicityassay, the % cell survival is plotted as a histogram in the order (leftto right): Cmpd 8a, 8b, 8c, 8d, 11 and 14, respectively.

FIGS. 9A-F depict fluorescence excitation and emission spectra of Cmpds27-31 and 33, respectively, in solution (solid lines) and in thepresence of Aβ peptide (dashed lines).

FIGS. 10A-F show fluorescence intensity versus concentration ofaggregated Ab peptides for Cmpds 27-31 and 33, respectively.

FIGS. 11A-F shows images of plaques that were stained with FIG. 11 A)compound 27, FIG. 11 B) compound 28, FIG. 11 C) compound 29, FIG. 11 D)compound 30, FIG. 11 E) compound 31, or FIG. 11 F) compound 33, asdescribed herein.

DETAILED DESCRIPTION I. Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “electron donating group (EDG)” refers to a chemical moietythat modifies the electrostatic forces acting on a nearby chemicalmoiety by donating negative charge to that chemical moiety. In someembodiments, the electron donating group donates negative charge to theπ-conjugation element of the compounds disclosed herein.

The term “π-conjugation element,” “πCE” or “pi-conjugation element”refers to a divalent chemical moiety that forms a π-conjugated systemthat has alternating single and multiple bonds (e.g., double bonds) suchthat the electrons in the p-orbitals of the atoms in the system aredelocalized. In some embodiments, the single and multiple bonds in theπ-conjugated element can be in a planar or substantially planarorientation.

The term “water soluble group” refers to a chemical moiety thatincreases the water solubility of the compounds to which it is attached.Increasing the water solubility can be measured using existingtechniques in the art, such as by determining a partition constant ofthe compounds with and without an attached water soluble group. In someembodiments, the partition constant can be measured by mixing a compoundwith water and a hydrophobic solvent, such as octanol. The morehydrophobic a compound, the higher its partition constant. The morehydrophilic a compound, the lower its partition constant. In someembodiments, the water soluble groups described herein can improve thewater solubility of precursor molecules by decreasing their partitioncoefficient. In some embodiments, the water soluble groups can decreasethe partition constant of precursor molecules (which have a higherpartition constant before attachment of the water soluble group) atleast by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In someembodiments, the water soluble groups described herein can decrease thepartition constant of precursor molecules by 1-fold, 2-fold, 3-fold,4-fold, or greater.

The term “amyloid” is used herein according to its customary meaning inthe art. Amyloids contain a plurality of associated amyloid peptides,such as aggregates of amyloid peptides. Thus, in some embodiments,amyloids include an amyloid peptide aggregated with one or more amyloidpeptides. In some embodiments, amyloids include “amyloid plaques,”amyloid deposits,” “amyloid aggregates” or “aggregates of amyloidpeptides.” The compounds described herein can associate with (e.g.,bind) an amyloid peptide and/or an amyloid. In certain embodiments, thecompounds described herein can associate with an amyloid by hydrophobicinteractions.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e. unbranched) or branched chain,or combination thereof, which may be fully saturated, mono- orpolyunsaturated and can include di- and multivalent radicals, having thenumber of carbon atoms designated (i.e. C₁-C₁₀ means one to tencarbons). Examples of saturated hydrocarbon radicals include, but arenot limited to, groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs andisomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and thelike. An unsaturated alkyl group is one having one or more double bondsor triple bonds. Examples of unsaturated alkyl groups include, but arenot limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy isan alkyl attached to the remainder of the molecule via an oxygen linker(—O—).

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkyl, as exemplified, but not limited,by —CH₂CH₂CH₂CH₂—, and further includes those groups described below as“heteroalkylene.” Typically, an alkyl (or alkylene) group will have from1 to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred. A “lower alkyl” or “lower alkylene” is a shorter chainalkyl or alkylene group, generally having eight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of atleast one carbon atoms and at least one heteroatom selected from thegroup consisting of O, N, P, Si and S. The nitrogen and sulfur atoms mayoptionally be oxidized, and the nitrogen heteroatom may optionally bequaternized. The heteroatom(s) O, N, P and S and Si may be placed at anyinterior position of the heteroalkyl group or at the position at whichthe alkyl group is attached to the remainder of the molecule. Examplesinclude, but are not limited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH-₂-CH₃, and —CN. Up to two heteroatoms maybe consecutive, such as, for example, —CH₂—NH—OCH₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)₂R′— represents both —C(O)₂R′—and —R′C(O)₂—. As described above, heteroalkyl groups, as used herein,include those groups that are attached to the remainder of the moleculethrough a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″″, —OR′, —SR′,and/or —SO₂R′. Where “heteroalkyl” is recited, followed by recitationsof specific heteroalkyl groups, such as —NR′R″ or the like, it will beunderstood that the terms heteroalkyl and —NR′R″ are not redundant ormutually exclusive. Rather, the specific heteroalkyl groups are recitedto add clarity. Thus, the term “heteroalkyl” should not be interpretedherein as excluding specific heteroalkyl groups, such as —NR′R″ or thelike.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl,and the like. Examples of heterocycloalkyl include, but are not limitedto, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is meant to include, but not be limited to,fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl,4-chlorobutyl, 3-bromopropyl, and the like.

The term “acyl” means C(O)R where R is a substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl or substituted orunsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together (i.e. afused ring aryl) or linked covalently. A fused ring aryl refers tomultiple rings fused together and at least one of the fused rings is anaryl ring. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S. Thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e. multiple rings fused together whereinat least one of the fused rings is a heteroaromatic ring). A 5,6-fusedring heteroarylene refers to two rings fused together, wherein one ringhas 5 members and the other ring has 6 members, and wherein at least onering is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylenerefers to two rings fused together, wherein one ring has 6 members andthe other ring has 6 members, and wherein at least one ring is aheteroaryl ring. And a 6,5-fused ring heteroarylene refers to two ringsfused together, wherein one ring has 6 members and the other ring has 5members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl,4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl,5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl,4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and6-quinolyl. Substituents for each of the above noted aryl and heteroarylring systems are selected from the group of acceptable substituentsdescribed below. An “arylene” and a “heteroarylene,” alone or as part ofanother substituent means a divalent radical derived from an aryl andheteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxyl)propyl, and the like).

The term “oxo” as used herein means oxygen that is double bonded to acarbon atom.

The term “alkylsulfonyl” as used herein means a moiety having theformula —S(O₂)—R′, where R′ is an alkyl group as defined above. R′ mayhave a specified number of carbons (e.g. “C₁-C₄ alkylsulfonyl”).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) are meant to include both substituted and unsubstitutedforms of the indicated radical. Preferred substituents for each type ofradical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compounddisclosed herein includes more than one R group, for example, each ofthe R groups is independently selected as are each R′, R″, R′ and R″″groups when more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: halogen, —OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl and substituted or unsubstitutedheteroaryl. When a compound disclosed herein includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′ and R″″ groups when more than one of these groups ispresent.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′— or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, orS(O)₂NR′—. The substituents R, R′, R″ and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted        alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,            unsubstituted alkyl, unsubstituted heteroalkyl,            unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,            unsubstituted aryl, unsubstituted heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            and heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, or heteroaryl, substituted with at least one                substituent selected from oxo, —OH, —NH₂, —SH, —CN,                —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstituted                heteroalkyl, unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, and unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein meansa group selected from all of the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl isa substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds disclosed herein containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds disclosed herein contain relativelybasic functionalities, acid addition salts can be obtained by contactingthe neutral form of such compounds with a sufficient amount of thedesired acid, either neat or in a suitable inert solvent. Examples ofpharmaceutically acceptable acid addition salts include those derivedfrom inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like (see, for example, Bergeet al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977,66, 1-19). Certain specific compounds disclosed herein contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

Thus, the compounds disclosed herein may exist as salts, such as withpharmaceutically acceptable acids. Examples of such salts includehydrochlorides, hydrobromides, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, tartrates (e.g., (+)-tartrates,(−) -tartrates or mixtures thereof including racemic mixtures),succinates, benzoates and salts with amino acids such as glutamic acid.These salts may be prepared by methods known to those skilled in theart.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the compounds disclosed herein can be in aprodrug form. Prodrugs of the compounds described herein are thosecompounds that readily undergo chemical changes under physiologicalconditions to provide the compounds disclosed herein. Additionally,prodrugs can be converted to the compounds disclosed herein by chemicalor biochemical methods in an ex vivo environment. For example, prodrugscan be slowly converted to the compounds disclosed herein when placed ina transdermal patch reservoir with a suitable enzyme or chemicalreagent.

Certain compounds disclosed herein can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are encompassed within thescope disclosed herein. Certain compounds disclosed herein may exist inmultiple crystalline or amorphous forms. In general, all physical formsare equivalent for the uses contemplated disclosed herein.

Certain compounds disclosed herein possess asymmetric carbon atoms(optical centers) or double bonds; the racemates, diastereomers,tautomers, geometric isomers and individual isomers. The compoundsdisclosed herein do not include those which are known in the art to betoo unstable to synthesize and/or isolate.

The compounds disclosed herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compoundsdisclosed herein, whether radioactive or not, are encompassed within thescope of the disclosure.

Where a substituent of a compound provided herein is “R-substituted”(e.g. R¹-substituted), it is meant that the substituent is substitutedwith one or more of the named R groups (e.g. R¹) as appropriate. In someembodiments, the substituent is substituted with only one of the named Rgroups.

The terms “treating” or “treatment” refers to any indicia of success inthe treatment or amelioration of an injury, pathology or condition,including any objective or subjective parameter such as abatement;remission; diminishing of symptoms or making the injury, pathology orcondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; making the final point of degeneration lessdebilitating; improving a patient's physical or mental well-being. Thetreatment or amelioration of symptoms can be based on objective orsubjective parameters; including the results of a physical examination,neuropsychiatric exams, and/or a psychiatric evaluation. For example,the certain methods presented herein successfully treat cancer bydecreasing the incidence of cancer, in inhibiting its growth and orcausing remission of cancer.

An “effective amount” is an amount of a compound described hereinsufficient to contribute to the treatment, prevention, or reduction of asymptom or symptoms of a disease, or to inhibit effects of an amyloidrelative to the absence of the compound. Where recited in reference to adisease treatment, an “effective amount” may also be referred to as a“therapeutically effective amount.” A “reduction” of a symptom orsymptoms (and grammatical equivalents of this phrase) means decreasingof the severity or frequency of the symptom(s), or elimination of thesymptom(s). A “prophylactically effective amount” of a drug is an amountof a drug that, when administered to a subject, will have the intendedprophylactic effect, e.g., preventing or delaying the onset (orreoccurrence) a disease, or reducing the likelihood of the onset (orreoccurrence) of a disease or its symptoms. The full prophylactic effectdoes not necessarily occur by administration of one dose, and may occuronly after administration of a series of doses. Thus, a prophylacticallyeffective amount may be administered in one or more administrations. An“activity decreasing amount,” as used herein, refers to an amount ofantagonist required to decrease the activity of an enzyme relative tothe absence of the antagonist. A “function disrupting amount,” as usedherein, refers to the amount of antagonist required to disrupt thefunction of an osteoclast or leukocyte relative to the absence of theantagonist.

II. Amyloid Binding Compounds

In one aspect, compounds that associate with an amyloid (or amyloids)and/or an amyloid peptide (or amyloid peptides) are provided. In someembodiments, the compound has the structure of Formula (I),

In Formula I, “EDG” is an electron donor group. “πCE” is a π-conjugationelement. “WSG” is a water soluble group.

In some embodiments, EDG is substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl, —OR²,—NR⁴C(O)R³, —CONR⁴R⁵, —NR⁴R⁵, —SR⁶, or —PR⁷R⁸. In some embodiments, EDGis substituted alkyl, substituted cycloalkyl, substituted heteroalkyl,substituted heterocycloalkyl, substituted aryl, substituted heteroaryl,—OR², —NR⁴C(O)R³, —CONR⁴R⁵, —NR⁴R⁵, —SR⁶, or —PR⁷R⁸. In someembodiments, EDG is R¹-substituted or unsubstituted alkyl,R¹-substituted or unsubstituted cycloalkyl, R¹-substituted orunsubstituted heteroalkyl, R¹-substituted or unsubstitutedheterocycloalkyl, R¹-substituted or unsubstituted aryl, R¹-substitutedor unsubstituted heteroaryl, —OR², —NR⁴C(O)R³, —CONR⁴R⁵, —NR⁴R⁵, —SR⁶,or —PR⁷R⁸. In some embodiments, EDG is R¹-substituted alkyl,R¹-substituted cycloalkyl, R¹-substituted heteroalkyl, R¹-substitutedheterocycloalkyl, R¹-substituted aryl, R¹-substituted heteroaryl, —OR²,—NR⁴C(O)R³, —CONR⁴R⁵, —NR⁴R⁵, —SR⁶, or —PR⁷R⁸.

In some embodiments, R¹ is halogen, —CN, —OR⁹, —CONR¹⁰R¹¹, —NR¹⁰R¹¹,—SR⁹, —SOR⁹, —SO₂R⁹, —COR⁹, —COOR⁹, —NR¹⁰COR⁹, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In some embodiments, R¹ is halogen, —CN, —OR⁹,—CONR¹⁰R¹¹, —NR¹⁰R¹¹, SR⁹, —SOR⁹, —SO₂R⁹, —COR⁹, —COOR⁹, —NR¹⁰COR⁹,R^(12a)-substituted or unsubstituted alkyl, R^(12a)-substituted orunsubstituted heteroalkyl, R^(12a)-substituted or unsubstitutedcycloalkyl, R^(12a)-substituted or unsubstituted heterocycloalkyl,R^(12a)-substituted or unsubstituted aryl, or R^(12a)-substituted orunsubstituted heteroaryl. In some embodiments, R¹ is halogen, —OR⁹,—NR¹⁰R¹¹, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl. In some embodiments, R¹ is —OR⁹, —NR¹⁰R¹¹,unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl. In some embodiments, when R¹ is attached toalkyl, cycloalkyl, or aryl, R¹ includes at least one heteroatom. In someembodiments, R¹ includes at least one heteroatom. In some embodiments,R¹ is —OR⁹ or —NR¹⁰R¹¹. In some embodiments, R¹ is —NR¹⁰R¹¹.

In certain embodiments, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independentlyhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. Incertain embodiments, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independentlyhydrogen, R¹²-substituted or unsubstituted alkyl, R¹²-substituted orunsubstituted heteroalkyl, R¹²-substituted or unsubstituted cycloalkyl,R¹²-substituted or unsubstituted heterocycloalkyl, R¹²-substituted orunsubstituted aryl or R¹²-substituted or unsubstituted heteroaryl. Insome embodiments, R⁴ and R⁵ are optionally joined together to form asubstituted or unsubstituted heterocycloalkyl, or substituted orunsubstituted heteroaryl. In some embodiments, R⁴ and R⁵ are optionallyjoined together to form R¹²-substituted or unsubstitutedheterocycloalkyl, or R¹²-substituted or unsubstituted heteroaryl.

In some embodiments, R⁹, R¹⁰ and R¹¹ are independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In some embodiments, R⁹, R¹⁹and R¹¹ are independently hydrogen, R¹²-substituted or unsubstitutedalkyl, R¹²-substituted or unsubstituted heteroalkyl, R¹²-substituted orunsubstituted cycloalkyl, R¹²-substituted or unsubstitutedheterocycloalkyl, R¹²-substituted or unsubstituted aryl, orR¹²-substituted or unsubstituted heteroaryl. In certain embodiments, R¹⁰and R¹¹ are optionally joined together to form an substituted orunsubstituted heterocycloalkyl, or substituted or unsubstitutedheteroaryl. In certain embodiments, R¹⁰ and R¹¹ are optionally joinedtogether to form an R¹²-substituted or unsubstituted heterocycloalkyl,or R¹²-substituted or unsubstituted heteroaryl.

R¹² and R^(12a) are independently halogen, —CN, —SR¹³, —SOR¹³, —SO₂R¹³,OR¹³, —NR¹⁴R¹⁵, —COR¹⁵, —COOR¹⁵, CONR¹⁴R¹⁵, —NR¹⁴COR¹⁵, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In some embodiments, R¹² and R^(12a) areindependently halogen, —CN, —SR¹³, —SOR¹³, —SO₂R¹³, —OR¹³, —NR¹⁴R¹⁵,—COR¹⁵, —COOR¹⁵, CON¹⁴R¹⁵, —NR¹⁴COR¹⁵, R¹⁶-substituted or unsubstitutedalkyl, R¹⁶-substituted or unsubstituted heteroalkyl, R¹⁶-substituted orunsubstituted cycloalkyl, R¹⁶-substituted or unsubstitutedheterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, orR¹⁶-substituted or unsubstituted heteroaryl. In some embodiments, R¹² is—OR¹³, —NR¹⁴R¹⁵, R¹⁶-substituted or unsubstituted alkyl, R¹⁶-substitutedor unsubstituted heteroalkyl, R¹⁶-substituted or unsubstitutedcycloalkyl, R¹⁶-substituted or unsubstituted heterocycloalkyl,R¹⁶-substituted or unsubstituted aryl, or R¹⁶-substituted orunsubstituted heteroaryl.

R¹³, R¹⁴ and R¹⁵ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In some embodiments, R¹³, R¹⁴ and R¹⁵ areindependently hydrogen R¹⁶-substituted or unsubstituted alkyl,R¹⁶-substituted or unsubstituted heteroalkyl, R¹⁶-substituted orunsubstituted cycloalkyl, R¹⁶-substituted or unsubstitutedheterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, orR¹⁶-substituted or unsubstituted heteroaryl. In some embodiments, R¹³,R¹⁴, and R¹⁵ are independently hydrogen, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted arylor unsubstituted heteroaryl. In some embodiments, R¹³, R¹⁴ and R¹⁵ areindependently hydrogen or unsubstituted alkyl.

R¹⁶ is halogen, —NH₂, —OH, —SH, —COOH, —COH, unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

In some embodiments, R¹² is —OR¹³ or —NR¹⁴R¹⁵. In some embodiments,R^(12a) is —OR¹³ or —NR¹⁴R¹⁵. In some embodiments, where R^(12a) formspart of an R¹ substituent (e.g. where R¹ is an alkyl, cycloalkyl oraryl), R^(12a) includes a heteroatom. In some embodiments, where R^(12a)forms part of an R¹ substituent (e.g. where R¹ is an alkyl, cycloalkylor aryl), R^(12a) is —OR¹³ or —NR¹⁴R¹⁵.

In some embodiments, R⁴ and R⁵ are independently hydrogen orR¹²-substituted or unsubstituted alkyl. In some embodiments, R⁴ and R⁵are independently hydrogen, R¹²-substituted or unsubstituted C₁-C₂₀(e.g., C₁-C₁₀) alkyl, or R¹²-substituted or unsubstituted heteroalkyl.In some embodiments, R⁴ and R⁵ are optionally joined together to form anR¹²-substituted or unsubstituted heterocycloalkyl. The R¹²-substitutedor unsubstituted heterocycloalkyl can be R¹²-substituted orunsubstituted piperidinyl, R¹²-substituted or unsubstituted morpholinyl,R¹²-substituted or unsubstituted tetrahydrofuranyl, R¹²-substituted orunsubstituted tetrahydrothienyl, or R¹²-substituted or unsubstitutedpiperazinyl. In some embodiments, R¹² is R¹⁶-substituted orunsubstituted C₁-C₂₀ (e.g., C₁-C₁₀) alkyl or R¹⁶-substituted orunsubstituted heteroalkyl. R¹⁶ can be unsubstituted C₄-C₈heterocycloalkyl.

In some embodiments, R⁴ and R⁵ are joined together to formR¹²-substituted or unsubstituted heteroaryl. The R¹²-substituted orunsubstituted heteroaryl can be R¹²-substituted or unsubstitutedpurinyl, R¹²-substituted or unsubstituted pyrimidinyl, R¹²-substitutedor unsubstituted imidazolyl, R¹²-substituted or unsubstitutedpyrrolopyridinyl (e.g., 1H-pyrrolo[2,3-b]pyridinyl), R¹²-substituted orunsubstituted pyrimidinyl, R¹²-substituted or unsubstituted indazolyl(e.g., 1H-indazolyl), or R¹²-substituted or unsubstitutedpyrrolopyrimidinyl (e.g., 7H-pyrrolo[2,3-d]pyrimidinyl). In someembodiments, R⁴ and R⁵ are joined together to form R¹²-substituted orunsubstituted pyrrolopyrimidinyl, R¹²-substituted or unsubstitutedindolyl, R¹²-substituted or unsubstituted pyrazolyl, R¹²-substituted orunsubstituted indazolyl, R¹²-substituted or unsubstituted imidazolyl,R¹²-substituted or unsubstituted thiazolyl, R¹²-substituted orunsubstituted benzothiazolyl, R¹²-substituted or unsubstituted oxazolyl,R¹²-substituted or unsubstituted benzimidazolyl, R¹²-substituted orunsubstituted benzoxazolyl, R¹²-substituted or unsubstituted isoxazolyl,R¹²-substituted or unsubstituted benzisoxazolyl, R¹²-substituted orunsubstituted triazolyl, R¹²-substituted or unsubstitutedbenzotriazolyl, R¹²-substituted or unsubstituted quinolinyl,R¹²-substituted or unsubstituted isoquinolinyl, R¹²-substituted orunsubstituted quinazolinyl, R¹²-substituted or unsubstitutedpyrimidinyl, R¹²-substituted or unsubstituted pyridinyl N-oxide,R¹²-substituted or unsubstituted furanyl, R¹²-substituted orunsubstituted thiophenyl, R¹²-substituted or unsubstituted benzofuranyl,R¹²-substituted or unsubstituted benzothiophenyl, R¹²-substituted orunsubstituted imidazopyridazinyl (e.g., imidazo[1,2b]pyridazinyl). Insome embodiments, R⁴ and R⁵ are joined together to form R¹²-substitutedor unsubstituted 6,5 fused ring heteroaryl, R¹²-substituted orunsubstituted 5,6 fused ring heteroaryl, R¹²-substituted orunsubstituted 5,5 fused ring heteroaryl, or R¹²-substituted orunsubstituted 6,6 fused ring heteroaryl. In other embodiments, R⁴ and R⁵are joined together to form a R¹²-substituted or unsubstituted 5 or 6membered heteroaryl having at least 2 (e.g. 2 to 4) ring nitrogens.

In some embodiments, the pi-conjugation element has the formula:-L¹-(A¹)_(q)-L²-(A²)_(r)-L³- or -L¹-(A¹)_(q)-L⁴-A³-L²-(A²)_(r)-L³-. L¹,L², L³ and L⁴ are independently a bond or a linking group having theformula:

In the formula above, the symbol x is an integer from 1 to 50. In someembodiments, x is an integer from 1 to 10, from 1 to 20, from 1 to 30,or from 1 to 40. In some embodiments, x is an integer from 1 to 3. Insome embodiments, x is an integer of 1. R^(17a) and R^(17b) areindependently hydrogen, halogen, —CN, —OR¹⁸, CONR¹⁹R²⁰, —NR¹⁹R²⁰, —SR¹⁸,—SOR¹⁸, —SO₂R¹⁸, —COR¹⁸, —COOR¹⁸, —NR¹⁹COR²⁰, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In some embodiments, R^(17a) and R^(17b) areindependently hydrogen, halogen, —CN, —OR¹⁸, —CONR¹⁹R²⁰, —NR¹⁹R²⁰, SR¹⁸,—SOR¹⁸, —SO₂R¹⁸, —COR¹⁸, —COOR¹⁸, —NR¹⁹COR²⁰, R²¹-substituted orunsubstituted alkyl, R²¹-substituted or unsubstituted heteroalkyl,R²¹-substituted or unsubstituted cycloalkyl, R²¹-substituted orunsubstituted heterocycloalkyl, R²¹-substituted or unsubstituted aryl,or R²¹-substituted or unsubstituted heteroaryl.

A¹, A² and A³ are independently substituted or unsubstituted arylene orsubstituted or unsubstituted heteroarylene. In some embodiments, A¹, A²and A³ are independently R¹⁷-substituted or unsubstituted arylene orR¹⁷-substituted or unsubstituted heteroarylene. The symbols q and r areindependently 0 or 1.

In some embodiments, the pi-conjugation element has the formula:-L¹-(A¹)_(q)-L²-(A²)_(r)-L³-. In certain embodiments, L¹ and L³ arebonds, L² is a linking group (as defined above or below), A¹ and A² aresubstituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene, and q and r are 1. In certain embodiments, L¹ and L³ arebonds, L² is a linking group (as defined above or below), A¹ and A² areR¹⁷-substituted or unsubstituted arylene, or R¹⁷-substituted orunsubstituted heteroarylene, and q and r are 1. In some embodiments, L¹,L² and L³ are bonds, A¹ and A² are R¹⁷-substituted or unsubstitutedarylene or R¹⁷-substituted or unsubstituted heteroarylene, and q is 1and r is 0.

In some embodiments, the pi-conjugation element has the formula:-L¹-(A¹)_(q)-L⁴-A³-L²-(A²)_(r)-L³-. In some embodiments, L¹ and L³ arebonds, L² and L⁴ are linking groups (as defined above or below), A¹, A²and A³ are substituted or unsubstituted arylene, or substituted orunsubstituted heteroarylene, and q and r are 1. In some embodiments, L¹and L³ are bonds, L² and L⁴ are linking groups (as defined above orbelow), A¹, A² and A³ are R¹⁷-substituted or unsubstituted arylene orR¹⁷-substituted or unsubstituted heteroarylene, and q and r are 1. Insome embodiments, the pi-conjugation element is substituted orunsubstituted arylene or substituted or unsubstituted heteroarylene. Insome embodiments, the pi-conjugation element is R¹⁷-substituted orunsubstituted arylene or R¹⁷-substituted or unsubstituted heteroarylene.In certain embodiments, the pi-conjugation element is substituted orunsubstituted phenylene or substituted or unsubstituted naphthylene. Incertain embodiments, the pi-conjugation element is R¹⁷-substituted orunsubstituted phenylene or R¹⁷-substituted or unsubstituted naphthylene.

In certain embodiments, compounds disclosed herein can exhibit increasedfluorescence when bound to amyloids. In certain embodiments, thepi-conjugation element is in a planar or substantially planarorientation when bound to an amyloid. In some embodiments, negativecharge donated from EDG can enhance the fluorescent properties of thecompounds herein and improve detection of amyloids (e.g., a amyloidplaque).

In some embodiments, a linking group (L¹, L², L³ and L⁴) has theformula:

The symbol x is an integer from 1 to 50. In some embodiments, x is aninteger from 1 to 10, from 1 to 20, from 1 to 30, or from 1 to 40. Insome embodiments, x is an integer from 1 to 5, 1 to 3, 2 or 1. In someembodiments, x is an integer from 1 to 3. In some embodiments, x is aninteger of 1. In some embodiments, L¹, L², L³ and L⁴ are independently abond.

In some embodiments, A¹, A² and A³ are independently substituted orunsubstituted arylene, or substituted or unsubstituted heteroarylene. Insome embodiments, A¹, A² and A³ are independently R¹⁷-substituted orunsubstituted arylene, or R¹⁷-substituted or unsubstitutedheteroarylene. In certain embodiments, q and r are independently 0 or 1.In some embodiments, q is 1 and r is 0. In some embodiments, q is 0 andr is 1. In some embodiments, A¹, A² and A³ are independently substitutedor unsubstituted phenylene, or substituted or unsubstituted naphthylene.In some embodiments, A¹, A² and A³ are independently R¹⁷-substituted orunsubstituted phenylene, or R¹⁷-substituted or unsubstitutednaphthylene. In some embodiments, A¹, A² and A³ are independentlyR¹⁷-substituted or unsubstituted phenylene. In some embodiments, A¹, A²and A³ are independently substituted or unsubstituted phenylene. In someembodiments, A¹, A² and A³ are independently substituted orunsubstituted naphthylene. In some embodiments, A¹, A² and A³ areindependently R¹⁷-substituted or unsubstituted naphthylene.

In some embodiments, R¹⁷ is independently halogen, —CN, —OR¹⁸,—CONR¹⁹R²⁰, —NR¹⁹R²⁰, —SR¹⁸, —SOR¹⁸, —SO₂R¹⁸, —COR¹⁸, —COOR¹⁸,—NR¹⁹COR²⁰, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. In someembodiments, R¹⁷ is independently halogen, —CN, —OR¹⁸, —CONR¹⁹R²⁰,—NR¹⁹R²⁰, —SR¹⁸, —SOR¹⁸, —SO₂R¹⁸, —COR¹⁸, —COOR¹⁸, —NR¹⁹COR²⁰,R²¹-substituted or unsubstituted alkyl, R-²¹substituted or unsubstitutedheteroalkyl, R²¹-substituted or unsubstituted cycloalkyl,R²¹-substituted or unsubstituted heterocycloalkyl, R²¹-substituted orunsubstituted aryl, or R²¹-substituted or unsubstituted heteroaryl. Insome embodiments, R¹⁷ is —OR¹⁸, —NR¹⁹R²⁰, R²¹⁻substituted orunsubstituted alkyl, R²¹-substituted or unsubstituted heteroalkyl,R²¹-substituted or unsubstituted cycloalkyl, R²¹-substituted orunsubstituted heterocycloalkyl, R²¹-substituted or unsubstituted aryl,or R²¹-substituted or unsubstituted heteroaryl. In some embodiments, R¹⁷is R²¹-substituted or unsubstituted C₁-C₂₀ (e.g., C₁-C₁₀) alkyl, orR²¹-substituted or unsubstituted heteroalkyl.

R¹⁸, R¹⁹ and R²⁰ are independently hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R¹⁸, R¹⁹ and R²⁰ are independently hydrogen,R²¹-substituted or unsubstituted alkyl, R²¹-substituted or unsubstitutedheteroalkyl, R²¹-substituted or unsubstituted cycloalkyl,R²¹-substituted or unsubstituted heterocycloalkyl, R²¹-substituted orunsubstituted aryl, or R²¹-substituted or unsubstituted heteroaryl. Insome embodiments, R²¹ is halogen, —OR²², —NR²³R²⁴, halogen, —CN, —OR²²,—CONR²³R²⁴, —NR²³R²⁴, —SR²², —SOR²², —SO₂R²², —COR²², —COOR²²,—NR²³COR²⁴, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl. R²¹ canbe halogen, —OR²², —NR²³R²⁴, halogen, —CN, —OR²², —CONR²³R²⁴, —NR²³R²⁴,—SR²², —SOR²², —SO₂R²², —COR²², —COOR²², —NR²³COR²⁴, R^(21a)-substitutedor unsubstituted alkyl, R^(21a)-substituted or unsubstitutedheteroalkyl, R^(21a)-substituted or unsubstituted cycloalkyl,R^(21a)-substituted or unsubstituted heterocycloalkyl,R^(21a)-substituted or unsubstituted aryl, or R^(21a)-substituted orunsubstituted heteroaryl. In some embodiments, R^(21a) is halogen, —NH₂,—OH, —SH, —COOH, —COH, unsubstituted alkyl, unsubstituted, heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl or unsubstituted heteroaryl. R²², R²³ and R²⁴ are independentlyhydrogen or unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstitutedaryl or unsubstituted heteroaryl. In some embodiments, R²², R²³ and R²⁴are independently hydrogen or unsubstituted alkyl.

In some embodiments, the water soluble group is substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In some embodiments, the water soluble groupis substituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, substituted aryl, or substitutedheteroaryl. In some embodiments, the water soluble group isR²⁵-substituted or unsubstituted alkyl, R²⁵-substituted or unsubstitutedheteroalkyl, R²⁵-substituted or unsubstituted cycloalkyl,R²⁵-substituted or unsubstituted heterocycloalkyl, R²⁵-substituted orunsubstituted aryl, R²⁵-substituted or unsubstituted heteroaryl. In someembodiments, the water soluble group is R²⁵-substituted alkyl,R²⁵-substituted heteroalkyl, R²⁵-substituted cycloalkyl, R²⁵-substitutedheterocycloalkyl, R²⁵-substituted aryl, R²⁵-substituted heteroaryl.

R²⁵ is halogen, —CN, —OR²⁶, —CONR²⁷R²⁸, —NR²⁷R²⁸, —SR²⁶, —SOR²⁶,—SO₂R²⁶, —COR²⁶, —COOR²⁶, —NR²⁷COR²⁸, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. In some embodiments, R²⁵ is halogen, —CN, —OR²⁶, —CONR²⁷R²⁸,—NR²⁷R²⁸, —SR²⁶, —SOR²⁶, —SO₂R²⁶, —COR²⁶, —COOR²⁶, —NR²⁷COR²⁸,R²⁹-substituted or unsubstituted alkyl, R²⁹-substituted or unsubstitutedheteroalkyl, R²⁹-substituted or unsubstituted cycloalkyl,R²⁹-substituted or unsubstituted heterocycloalkyl, R²⁹-substituted orunsubstituted aryl, or R²⁹-substituted or unsubstituted heteroaryl. Insome embodiments, R²⁶, R²⁷ and R²⁸ are independently hydrogen,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In some embodiments, R²⁶, R²⁷and R²⁸ are independently hydrogen, R²⁹-substituted or unsubstitutedalkyl, R²⁹-substituted or unsubstituted heteroalkyl, R²⁹-substituted orunsubstituted cycloalkyl, R²⁹-substituted or unsubstitutedheterocycloalkyl, R²⁹-substituted or unsubstituted aryl, orR²⁹-substituted or unsubstituted heteroaryl. In certain embodiments, R²⁷and R²⁸ are optionally joined together to form a substituted orunsubstituted heterocycloalkyl, or a substituted or unsubstitutedheteroaryl. In certain embodiments, R²⁷ and R²⁸ are optionally joinedtogether to form a R²⁹-substituted or unsubstituted heterocycloalkyl, ora R²⁹-substituted or unsubstituted heteroaryl.

R²⁹ is halogen, —CN, —OR³⁰, —CONR³¹R³², —NR³¹R³², —SR³⁰, —SOR³⁰,—SO₂R³⁰, —COR³⁰, —COOR³⁰, —NR³¹COR³², substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl. In some embodiments, R²⁹ is unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. Insome embodiments, R³⁰, R³¹ and R³² are independently hydrogen orsubstituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. In some embodiments, R³⁰, R³¹and R³² are independently hydrogen or unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl or unsubstituted heteroaryl. In some embodiments,R³⁰, R³¹ and R³² are independently hydrogen or unsubstituted alkyl.

In some embodiments, the water soluble group can include a moiety thatincreases the water solubility of a molecule. In some embodiments, thewater soluble group can include a moiety containing a heteroatom (e.g.,oxygen). In some embodiments, the heteroatom can be oxygen or nitrogen.

In some embodiments, the water soluble group is an ethylene glycolmoiety having the formula:

In some embodiments, y is an integer from 1 to 50. In some embodiments,y is an integer from 1 to 10, from 1 to 20, from 1 to 30, or from 1 to40. In some embodiments, R²⁹ is —OMe.

In some embodiments, the water soluble group is R²⁹-substituted orunsubstituted C₁-C₂₀ (e.g., C₁-C₁₀) alkyl or R²⁹-substituted orunsubstituted heteroalkyl. In some embodiments, R²⁹ is —OH. In someembodiments, the water soluble group can be —(CH₂)_(b)—(CH₂OH)—CH₂OH,and b is an integer from 0 to 20, or from 0-10.

In some embodiments, the compound has the structure:

In Formula IIa, q and r are independently 0 or 1, and y is an integerfrom 1 to 10. L¹, L², L³, A¹, A², R⁴, R⁵ and R²⁹ are as defined above.

In some embodiments, the compound has the structure:

In Formula IIb, q and r are independently 0 or 1, and y is an integerfrom 1 to 10. L¹, L², L³, L⁴, A¹, A², A³, R⁴, R⁵ and R²⁹ are as definedabove.

In some embodiments, the compound has the structure:

In Formula IIIa, m is an integer from 0 to 4, z is an integer from 0 to4, and y is an integer from 1 to 10. L¹, L², L³, R⁴, R⁵, R¹⁷ and R²⁹ areas defined above.

In some embodiments, the compound has the structure:

In Formula IIIb, m is an integer from 0 to 6, z is an integer from 0 to6, and y is an integer from 1 to 10. L¹, L², L³, R⁴, R⁵, R¹⁷ and R²⁹ areas defined above. In some embodiments, m is 0. In some embodiments, z is0. In some embodiments, m is 1. In some embodiments, z is 1.

In some embodiments, the compound has the structure:

In Formula IVa, y is an integer from 1 to 10, and z is an integer from 0to 4. R⁴, R⁵, R¹⁷ and R²⁹ are as defined above. In some embodiments, R²⁹is —OMe.

In some embodiments, the compound has the structure:

In Formula IVb, y is an integer from 1 to 10, and z is an integer from 0to 6. R⁴, R⁵, R¹⁷ and R²⁹ are as defined above. In some embodiments, R²⁹is —OMe. In some embodiments, m is 0. In some embodiments, z is 0. Insome embodiments, m is 1. In some embodiments, z is 1.

In some embodiments, the compound has the structure:

In Formula IVc, m is an integer from 0 to 4, x is an integer from 1 to10, y is an integer from 1 to 10, and z is an integer from 0 to 4. Insome embodiments, x is 1, and m and z are 0. R⁴, R⁵, R¹⁷ and R²⁹ are asdefined above. In some embodiments, R²⁹ is —OMe.

In some embodiments, each substituted group described above in thecompounds of the Formulae provided herein is substituted with at leastone substituent group. More specifically, in some embodiments, eachsubstituted alkyl, substituted heteroalkyl, substituted cycloalkyl,substituted heterocycloalkyl, substituted aryl, substituted heteroaryl,substituted alkylene, substituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene described above in thecompounds of the Formulae provided herein is substituted with at leastone substituent group. In other embodiments, at least one or all ofthese groups are substituted with at least one size-limited substituentgroup. Alternatively, at least one or all of these groups aresubstituted with at least one lower substituent group.

In other embodiments of the compounds of the Formulae provided herein,each substituted or unsubstituted alkyl is a substituted orunsubstituted C₁-C₂₀ alkyl, each substituted or unsubstitutedheteroalkyl is a substituted or unsubstituted 2 to 20 memberedheteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₄-C₈ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8membered heterocycloalkyl, each substituted or unsubstituted alkylene isa substituted or unsubstituted C₁-C₂₀ alkylene, each substituted orunsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20membered heteroalkylene, each substituted or unsubstituted cycloalkylenesubstituted or unsubstituted C₄-C₈ cycloalkylene, and each substitutedor unsubstituted heterocycloalkylene is a substituted or unsubstituted 4to 8 membered heterocycloalkylene.

Alternatively, each substituted or unsubstituted alkyl is a substitutedor unsubstituted C₁-C₈ alkyl, each substituted or unsubstitutedheteroalkyl is a substituted or unsubstituted 2 to 8 memberedheteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl, each substituted or unsubstituted alkylene isa substituted or unsubstituted C₁-C₈ alkylene, each substituted orunsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8membered heteroalkylene, each substituted or unsubstituted cycloalkylenesubstituted or unsubstituted C₅-C₆ cycloalkylene, and each substitutedor unsubstituted heterocycloalkylene is a substituted or unsubstituted 5to 7 membered heterocycloalkylene.

In some embodiments, the compounds of the Formulae provided herein areone or more of the compounds set forth in Table 1 below:

TABLE 1 Compounds. Cmpd Structure 8a

8b

8c

8d

11

14

19

29

28

30

31

33

III. Pharmaceutical Compositions

In another aspect, pharmaceutical compositions disclosed herein (i.e.,formulations) can include a compound described herein in combinationwith a pharmaceutically acceptable excipient (e.g., carrier). Thepharmaceutical compositions include optical isomers, diastereomers, orpharmaceutically acceptable salts of the inhibitors disclosed herein.For example, in some embodiments, the pharmaceutical compositionsinclude a compound disclosed herein and citrate as a pharmaceuticallyacceptable salt. The compound included in the pharmaceutical compositionmay be covalently attached to a carrier moiety, as described above.Alternatively, the compound included in the pharmaceutical compositionis not covalently linked to a carrier moiety.

A “pharmaceutically acceptable carrier,” as used herein refers topharmaceutical excipients, for example, pharmaceutically,physiologically, acceptable organic or inorganic carrier substancessuitable for enteral or parenteral application that do not deleteriouslyreact with the active agent. Suitable pharmaceutically acceptablecarriers include water, salt solutions (such as Ringer's solution),alcohols, oils, gelatins, and carbohydrates such as lactose, amylose orstarch, fatty acid esters, hydroxymethycellulose, and polyvinylpyrrolidine. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds disclosed herein.

The compounds disclosed herein can be administered alone or can becoadministered to the subject. Coadministration is meant to includesimultaneous or sequential administration of the compounds individuallyor in combination (more than one compound). The preparations can also becombined, when desired, with other active substances (e.g. to reducemetabolic degradation).

A. Formulations

The compounds can be prepared and administered in a wide variety oforal, parenteral, and topical dosage forms. Thus, the compoundsdisclosed herein can be administered by injection (e.g. intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally). Also, the compounds described herein can beadministered by inhalation, for example, intranasally. Additionally, thecompounds disclosed herein can be administered transdermally. It is alsoenvisioned that multiple routes of administration (e.g., intramuscular,oral, transdermal) can be used to administer the compounds disclosedherein. Accordingly, pharmaceutical compositions can include apharmaceutically acceptable carrier or excipient and one or morecompounds disclosed herein.

For preparing pharmaceutical compositions from the compounds disclosedherein, pharmaceutically acceptable carriers can be either solid orliquid. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. A solidcarrier can be one or more substance that may also act as diluents,flavoring agents, binders, preservatives, tablet disintegrating agents,or an encapsulating material.

In powders, the carrier is a finely divided solid in a mixture with thefinely divided active component. In tablets, the active component ismixed with the carrier having the necessary binding properties insuitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from 5% to 70% of the activecompound. Suitable carriers are magnesium carbonate, magnesium stearate,talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoabutter, and the like. The term “preparation” is intended to include theformulation of the active compound with encapsulating material as acarrier providing a capsule in which the active component with orwithout other carriers, is surrounded by a carrier, which is thus inassociation with it. Similarly, cachets and lozenges are included.Tablets, powders, capsules, pills, cachets, and lozenges can be used assolid dosage forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

When parenteral application is needed or desired, particularly suitableadmixtures for the compounds disclosed herein are injectable, sterilesolutions, preferably oily or aqueous solutions, as well as suspensions,emulsions, or implants, including suppositories. In particular, carriersfor parenteral administration include aqueous solutions of dextrose,saline, pure water, ethanol, glycerol, propylene glycol, peanut oil,sesame oil, polyoxyethylene-block polymers, and the like. Ampoules areconvenient unit dosages. The compounds disclosed herein can also beincorporated into liposomes or administered via transdermal pumps orpatches. Pharmaceutical admixtures suitable for use herein include thosedescribed, for example, in Pharmaceutical Sciences (17th Ed., Mack Pub.Co., Easton, Pa.) and WO 96/05309, the teachings of both of which arehereby incorporated by reference.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

Also included are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to1000 mg, most typically 10 mg to 500 mg, according to the particularapplication and the potency of the active component. The compositioncan, if desired, also contain other compatible therapeutic agents.

Some compounds may have limited solubility in water and therefore mayrequire a surfactant or other appropriate co-solvent in the composition.Such co-solvents include: Polysorbate 20, 60, and 80; Pluronic F-68,F-84, and P-103; cyclodextrin; and polyoxyl 35 castor oil. Suchco-solvents are typically employed at a level between about 0.01% andabout 2% by weight.

Viscosity greater than that of simple aqueous solutions may be desirableto decrease variability in dispensing the formulations, to decreasephysical separation of components of a suspension or emulsion offormulation, and/or otherwise to improve the formulation. Such viscositybuilding agents include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methyl cellulose, hydroxy propyl methylcellulose,hydroxyethyl cellulose, carboxymethyl cellulose, hydroxy propylcellulose, chondroitin sulfate and salts thereof, hyaluronic acid andsalts thereof, and combinations of the foregoing. Such agents aretypically employed at a level between about 0.01% and about 2% byweight.

The compositions disclosed herein may additionally include components toprovide sustained release and/or comfort. Such components include highmolecular weight, anionic mucomimetic polymers, gelling polysaccharides,and finely-divided drug carrier substrates. These components arediscussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841;5,212,162; and 4,861,760. The entire contents of these patents areincorporated herein by reference in their entirety for all purposes.

B. Effective Dosages

Pharmaceutical compositions provided herein include compositions inwhich the active ingredient is contained in a therapeutically effectiveamount, i.e., in an amount effective to achieve its intended purpose.The actual amount effective for a particular application will depend,inter alia, on the condition being treated. For example, whenadministered in methods to treat cancer, such compositions will containan amount of active ingredient effective to achieve the desired result(e.g. decreasing the number of cancer cells in a subject).

The dosage and frequency (single or multiple doses) of compoundadministered can vary depending upon a variety of factors, includingroute of administration; size, age, sex, health, body weight, body massindex, and diet of the recipient; nature and extent of symptoms of thedisease being treated; presence of other diseases or otherhealth-related problems; kind of concurrent treatment; and complicationsfrom any disease or treatment regimen. Other therapeutic regimens oragents can be used in conjunction with the methods and compoundsdisclosed herein.

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays, as known in theart.

Therapeutically effective amounts for use in humans may be determinedfrom animal models. For example, a dose for humans can be formulated toachieve a concentration that has been found to be effective in animals.

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient shouldbe sufficient to affect a beneficial therapeutic response in the patientover time. The size of the dose also will be determined by theexistence, nature, and extent of any adverse side effects. Generally,treatment is initiated with smaller dosages, which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached. In one embodiment, the dosage range is 0.001% to 10% w/v. Inanother embodiment, the dosage range is 0.1% to 5% w/v.

Dosage amounts and intervals can be adjusted individually to providelevels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

Utilizing the teachings provided herein, an effective prophylactic ortherapeutic treatment regimen can be planned that does not causesubstantial toxicity and yet is entirely effective to treat the clinicalsymptoms demonstrated by the particular patient. This planning shouldinvolve the careful choice of active compound by considering factorssuch as compound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration, and the toxicity profile of the selected agent.

C. Toxicity

The ratio between toxicity and therapeutic effect for a particularcompound is its therapeutic index and can be expressed as the ratiobetween LD₅₀ (the amount of compound lethal in 50% of the population)and ED₅₀ (the amount of compound effective in 50% of the population).Compounds that exhibit high therapeutic indices are preferred.Therapeutic index data obtained from cell culture assays and/or animalstudies can be used in formulating a range of dosages for use in humans.The dosage of such compounds preferably lies within a range of plasmaconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. See, e.g. Fingl etal., In: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Ch. 1, p. 1, 1975.The exact formulation, route of administration, and dosage can be chosenby the individual physician in view of the patient's condition and theparticular method in which the compound is used.

IV. Methods of Use

In one aspect, there is provided methods of detecting an amyloid peptideand/or amyloid. The methods of detection can employ spectroscopic (i.e.,UV-visible, fluorescence, and the like), radiographic, and otherdetection methods known in the art. In one embodiment, the methodsincludes contacting a compound as described herein with an amyloid,thereby forming a detectable amyloid complex, and detecting thedetectable amyloid complex, as described herein and known in the art. An“amyloid complex” is referred to herein as a complex of a compounddescribed herein and at least one amyloid peptide (e.g., an aggregate ofamyloid peptides). The compounds described herein can form a complexwith an amyloid by a variety of interactions, such as non-covalentinteractions (e.g., hydrophobic interactions or hydrogen bonding).

Amyloids described herein can be composed of at least one amyloidpeptide molecule. An amyloid peptide is referred to herein as a peptideor protein that can forms part of or is capable of forming an amyloid inassociation with other peptides or proteins. Amyloids described hereincan be composed of any amyloid peptide or amyloid protein that is knownto form amyloids. In some embodiments, amyloids include a plurality ofamyloid peptides and/or amyloid peptide molecules. In some embodiments,an amyloid includes an amyloid peptide molecule aggregated with one ormore amyloid peptide molecules.

In some embodiments, the amyloid peptides can include Aβ peptide, prionprotein, α-synuclein, or superoxide dismutase. In some embodiments, theamyloid peptides can include a portion of or functional fragment thereofof Aβ peptide, prion protein, α-synuclein, or superoxide dismutase. Insome embodiments, an amyloid peptide and/or amyloid can be solvated insolution and bound to one or more of the compounds described herein. Insome embodiments, the amyloids include amyloid peptides that arearranged in β-sheets that can allow for binding of the compoundsdescribed herein. In certain embodiments, the compounds described hereinexhibit increased fluorescence (as compared to free solution) when boundand interacting with amyloids via hydrophobic interactions.

In another aspect, the methods provided herein include methods ofassaying the compounds herein to detect binding of the compounds toamyloids and/or amyloid peptides. Using techniques known in the art andthe guidance provided herein, candidate compounds may be easily assayedfor their ability to bind to amyloids. For example, amyloid bindingagents having the structure of the Formulae provided herein orembodiments thereof may be assayed using in vitro assays. In someembodiments, in vitro assays can include measuring fluorescence of thecompounds herein when they are bound to amyloids versus in free solutionnot bound to the amyloids. Generally, an increase in fluorescenceindicates binding to an amyloid. Binding constants for the compoundsherein can also be determined using techniques known in the art. Forexample, competitive binding studies can be used to determine theeffectiveness of the compounds described herein for inhibiting, e.g.,IgG-Aβ peptide interactions. Cellular assays may also be used to assessthe binding properties of candidate amyloid binding agents having thestructure of the Formulae provided herein or embodiments thereof.Cellular assays include cells from any appropriate source, includingplant and animal cells (such as mammalian cells). The cellular assaysmay also be conducted in human cells. The selection of appropriate assaymethods is well within the capabilities of those having ordinary skillin the art.

Once compounds are identified that are capable of binding amyloids invitro and/or in a cell, the compounds may be further tested for theirability to selectively bind amyloid (e.g., in amyloid plaques) in animalmodels (e.g. whole animals, animal organs, or animal tissues). Thus, thecompounds described herein may be further tested in cell models oranimal models for their ability to cause detectable changes in phenotyperelated to a particular amyloid peptide and/or amyloid. In addition tocell cultures, animal models may be used to test the compounds describedherein for their ability to treat, for example, diseases associated withamyloids in an animal model. In some embodiments, the compoundsdescribed herein can be used to image amyloids in animal tissue. In someembodiments, the animal tissue is human tissue.

In a further aspect, there is provided a method of treating a diseaseassociated with amyloid peptides and/or amyloids in a subject in need ofsuch treatment. In some embodiments, the disease can be characterized byan accumulation of amyloids (e.g., amyloid plaques) in a subject. Themethods can include administering to the subject an effective amount(e.g. a therapeutically effective amount) of a compound having thestructure of the Formulae provided herein (or an embodiment thereof asdescribed above).

The term “subject” as used herein refers to a mammal to which apharmaceutical composition or formulation is administered. Exemplarysubjects include humans, as well as veterinary and laboratory animalssuch as horses, pigs, cattle, dogs, cats, rabbits, rats, mice, andaquatic mammals. In some embodiments, the subject is a human.

In some embodiments, the disease can include Alzheimer's disease, bovinespongiform encephalopathy (BSE), Parkinson's disease, Huntington'sdisease, Down's Syndrome, Dementia with Lewy Body, or AmyotrophicLateral Sclerosis (ALS). In some embodiments, the amyloid peptide is Aβpeptide and the disease is Alzheimer's disease. In some embodiments, themethods of treating described herein include a method of treatingAlzheimer's disease. In some embodiments, the methods of treatingdescribed herein include a method of treating Parkinson's disease.

Each patent, published patent application, and reference cited herein ishereby incorporated herein in its entirety and for all purposes.

V. Examples Example 1

General Procedure for the Preparation of Compounds Described Herein andin Examples 1a-1m.

To a round bottom flask containing a solution of aldehyde (5.0 mmol) and2-(2-(2-methoxyethoxy) ethoxy)ethyl 2-cyanoacetate (5.5 mmol) in 20 mlof THF was added 0.50 mmol of piperidine and the mixture was heated at50° C. The reaction was monitored by TLC and was completed within 21hours. The crude mixture was concentrated under reduced pressure and theproduct was purified via flash chromatography (10-30% ethyl acetate inhexane).

General Notes:

All the reagents were obtained (Aldrich, Acros) at highest commercialquality and used without further purification except where noted. Air-and moisture-sensitive liquids and solutions were transferred viasyringe or stainless steel cannula. Organic solutions were concentratedby rotary evaporation below 45° C. at approximately 20 mmHg. Allnon-aqueous reactions were carried out under anhydrous conditions.Yields refer to chromatographically and spectroscopically (1H NMR, 13CNMR) homogeneous materials, unless otherwise stated. Reactions weremonitored by thin-layer chromatography (TLC) carried out on 0.25 mm EMerck silica gel plates (60E-254) and visualized under UV light and/ordeveloped by dipping in solutions of 10% ethanolic phosphomolybdic acid(PMA) or p-anisaldehyde and applying heat. E. Merck silica gel (60,particle size 0.040-0.063 mm) was used for flash chromatography.Preparative thin-layer chromatography separations were carried out on0.25 or 0.50 mm E Merck silica gel plates (60E-254). NMR spectra wererecorded on Varian Mercury 300 or 400 MHz instruments and calibratedusing the residual undeuterated solvent as an internal reference. Thefollowing abbreviations were used to explain the multiplicities:s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, b=broad. Highresolution mass spectra (HRMS) were recorded on a VG 7070 HS massspectrometer under electron spray ionization (ESI) or electron impact(EI) conditions. Fluorescence spectroscopy data were recorded on aMD-5020 Photon Technology International Spectrophotometer at 25° C.

Important to the synthesis of compounds described herein was aKnoevenagel condensation of 1 equivalent of the appropriate aldehyde,e.g. 6, with 1.1 equivalents of the appropriate malonic acid derivative,e.g. 7. See Scheme 1. This reaction was catalyzed by piperidine (10%)and was completed within 21 hours in refluxing THF. See X. H. Chen, Z.J. Zhao, Y. Liu, P. Lu, Y. G. Wang, Chemistry Letters 2008, 37:570-571;M. A. Haidekker, T. P. Brady, D. Lichlyter, E. A. Theodorakis, Journalof the American Chemical Society 2006, 128:398-399. After a standardchromatographic purification on silica gel, the desired product 8 wasisolated in excellent yields (Table 2). Reagents and conditions forScheme 1: (a) 1.0 equiv 6, 1.1 equiv 7, 0.1 equiv piperidine, THF, 50°C., 21 h.

TABLE 2 Structures and yields for Cmpds 8a-8d. Comp. No R⁴ R⁵ Yield (%)8a Me H 98 8b Me OMe 98 8c Et H 90 8d nBu H 78

Naphthalene-based Cmpd 11 was synthesized by treatment of commerciallyavailable methoxy naphthaldehyde 9 with eight equivalents of lithiatedpiperidine and Knoevenagel condensation of the resulting aldehyde 10with cyano ester 7 (Scheme 2, 29% combined yield). See H. M. Guo, F.Tanaka, J. Org. Chem. 2009, 74:2417-2424. Scheme 2 reagents andconditions: (a) 8.0 equiv piperidine in benzene/HMPA: 1/1, 0° C., 8.0equiv nBuLi, 0° C., 15 min, then 1.0 equiv 9, 25° C., 12 h, 35%; (b) 1.0equiv 10, 1.1 equiv 7, 0.1 equiv piperidine, THF, 50° C., 21 h, 82%. Insome embodiments, R⁴, R⁵ and R¹⁶ can correspond to the R⁴, R⁵ and R¹⁶described above.

Cmpd 14 was prepared by condensation of aldehyde 6a with α-cyano ester12, followed by an acid-catalyzed deprotection of the acetonide unit(Scheme 3, 68% combined yield). See M. A. Haidekker, T. P. Brady, S. H.Chalian, W. Akers, D. Lichlyter, E. A. Theodorakis, Bioorg. Chem. 2004,32:274-289. Scheme 3 reagents and conditions: (a) 1.0 equiv 6a, 1.1equiv 12, 0.1 equiv piperidine, THF, 50° C., 21 h, 91%; (b) 1.5 mmol 13,0.10 g DOWEX-H+, 1:1 THF/MeOH, 25° C., 20 h, 75%.

Stilbene-based Cmpd 19 was synthesized in four steps that included: (a)conversion of benzyl bromide 15 to phosphonate 16; (b) Horner-Emmonsolefination of 16 with aldehyde 6a to form 17; (c) lithiation of bromide17 and formylation to produce aldehyde 18; and (d) Knoevenagelcondensation of the resulting aldehyde 18 with cyano ester 7 (Scheme 4,42% combined yield). See H. Meier, E. Karpuk, H. C. Holst, Eur. J. Org.Chem. 2006, 2609-2617; L. Viau, O. Maury, H. Le Bozec, Tetrahedron Lett.2004, 45:125-128. Scheme 4 reagents and conditions: (a) 1.0 equiv 15, 15equiv triethyl phosphite, 90° C., 19 h, 98%; (b) 1.0 equiv 16, 1.0 equivNaOMe, 1.0 equiv 6a, excess DMF, 25° C., 24 h, 74%; (c) 1.0 equiv 17,1.0 equiv n-BuLi, 1.33 equiv DMF, THF, −78° C., 60%; (d) 1.0 equiv 18,1.1 equiv 7, 0.1 equiv piperidine, THF, 50° C., 21 h, 97%. R¹ and R² inScheme 4 are specific for Scheme 4 and are not intended to correspond toR¹ and R² described above.

Example 1a (E)-2-(2-(2-methoxyethoxyl)ethoxy)ethyl2-cyano-3-(4-(dimethylamino)phenyl)acrylate (8a)

98%; yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 8.07 (s, 1H), 7.93 (d, 2H,J=9.0 Hz), 6.69 (d, 2H, J=9.1 Hz), 4.41 (m, 2H), 3.81-3.79 (m, 2H),3.73-3.65 (m, 6H), 3.56-3.54 (m, 2H), 3.37 (s, 3H), 3.10 (s, 6H); ¹³CNMR (100 MHz, CDCl₃) δ 164.2, 154.7, 153.6, 134.1, 119.3, 117.4, 111.4,93.6, 71.9, 70.8, 70.6, 70.5, 68.9, 65.0, 59.0, 40.0; HRMS Calc forC₁₉H₂₆N₂O₅(M)⁺ 362.1836 found 362.1841.

Example 1b(E)-2-(2-(2-methoxyethoxyl)ethoxy)ethyl2-cyano-3-(4-(dimethylamino)-2-methoxyphenyl)acrylate(8b)

98% yield; yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 8.64 (s, 1H), 8.39(d, 1H, J=9.2 Hz), 6.63 (dd, 1H, J=2.3 Hz, J=9.2 Hz), 6.01 (s, 1H), 4.40(m, 2H), 3.87 (s, 3H), 3.81-3.78 (m, 2H), 3.73-3.65 (m, 6H), 3.56-3.53(m, 2H), 3.36 (s, 3H), 3.10 (s, 6H); ¹³C NMR (400 MHz, CDCl₃) δ 165.0,162.2, 155.9, 148.5, 131.3, 118.4, 109.7, 105.4, 93.0, 92.0, 72.2, 71.1,70.9, 70.8, 69.2, 65.1, 59.3, 55.6, 40.4; HRMS Calc forC₂₀H₂₈N₂O₆(M+Na)⁺ 415.1840 found 415.1836.

Example 1c (Z)-2-(2-(2-methoxyethoxyl)ethoxy)ethyl2-cyano-3-(4-(diethylamino)phenyl)acrylate (8c)

90% yield; orange liquid; ¹H NMR (400 MHz, CDCl₃) δ 8.05 (s, 1H), 7.92(d, 2H, J=9.1 Hz), 6.67 (d, 2H, J=9.2 Hz), 4.42 (m, 2H), 3.82-3.79 (m,2H), 3.73-3.72 (m, 2H), 3.69-3.65 (m, 4H), 3.57-3.54 (m, 2H), 3.45 (q,4H, J=7.1 Hz), 3.37 (s, 3H), 1.23 (t, 6H, J=7.1 Hz); ¹³CNMR (100 MHz,CDCl₃) δ 164.7, 154.8, 151.9, 134.8, 119.0, 117.8, 111.4, 93.0, 72.2,71.1, 70.9, 70.8, 69.2, 65.2, 59.3, 45.0, 12.8; HRMS Calc forC₂₁H₃N₂O₅(M+Na)⁺ 413.2047 found 413.2053.

Example 1d(Z)-2-(2-(2-methoxyethoxyl)ethoxy)ethyl-2-cyano-3-(4-(dibutylamino)phenyl)acrylate(8d)

78% yield; yellow liquid; ¹HNMR (400 MHz, CDCl₃) δ 8.00 (s, 1H), 7.87(d, 2H, J=9.0 Hz), 6.60 (d, 2H, J=9.2 Hz), 4.38 (m, 2H), 3.78-3.76 (m,2H), 3.71-3.69 (m, 2H), 3.66-3.62 (m, 4H), 3.53-3.51 (m, 2H), 3.34-3.30(m, 7H), 1.57 (m, 4H), 1.34 (m, 4H), 0.94 (t, 6H, J=7.3 Hz); ¹³CNMR (100MHz, CDCl₃) δ 164.7, 154.7, 152.2, 134.6, 118.9, 117.9, 111.5, 92.8,72.1, 71.0, 70.8, 69.1, 65.2, 59.2, 51.1, 29.5, 20.4, 14.1; HRMS Calcfor C₂₅H₃₈N₂O₅(M+Na)⁺ 469.2673 found 469.2677.

Example 1e 6-(piperidin-1-yl)-2-naphthaldehyde (10)

To a 50 ml round bottom flask containing benzene (3 mL), HMPA (3 mL) andpiperidine (1.65 ml, 16.7 mmol) n-BuLi (1.6 M in hexane, 10.4 mL, 16.7mmol) was added via syringe, at 0° C. After stirring for 15 min, thereaction mixture was treated with a solution of6-methoxy-2-naphthaldehyde (390 mg, 2.09 mmol) in benzene: HMPA 1:1 (2ml). The reaction mixture was warmed to room temperature, left stirringfor 12 hours and then it was poured into cold 5% aqueous NaCl (30 ml).The mixture was extracted with diethyl ether (3×20 mL), dried over MgSO₄and concentrated. The product was purified via flash chromatography (20%EtOAc in hexanes) to give compound 9. 9: 35% yield, yellow solid; ¹H NMR(300 MHz, CDCl₃) δ 10.02 (s, 1H), 8.14 (s, 1H), 7.88-7.73 (m, 2H), 7.67(d, 1H, J=8.6 Hz), 7.32 (dd, 1H, J=2.5 Hz, J=9.1 Hz), 7.08 (d, 1H, J=2.4Hz), 3.42-3.32 (m, 4H), 1.85-1.57 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ192.2, 152.2, 138.8, 134.7, 131.6, 130.7, 127.5, 126.5, 123.6, 119.7,109.0, 49.8, 25.8, 24.6; HRMS calc for C₁₆H₁₇NO (M+H)⁺ 240.1383 found240.1387.

Example 1f(E)-2-(2-(2-methoxyethoxyl)ethoxy)ethyl-2-cyano-3-(6-(piperidin-1-yl)naphthalen-2-yl)acrylate(11)

82% yield; red liquid; ¹H NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H), 8.22 (d,1H, J=1.2 Hz), 8.10 (dd, 1H, J=1.8 Hz, J=8.8 Hz), 7.76 (d, 1H, J=9.2Hz), 7.65 (d, 1H, J=8.8 Hz), 7.29 (dd, 1H, J=2.4 Hz, J=9.2 Hz), 7.05 (d,1H, J=2.2 Hz), 4.47 (m, 2H), 3.85-3.82 (m, 2H), 3.74-3.66 (m, 6H),3.57-3.54 (m, 2H), 3.42-3.38 (m, 4H), 3.37 (s, 3H), 1.74-1.67 (m, 6H);¹³C NMR (100 MHz, CDCl₃) δ 163.4, 155.5, 151.9, 137.8, 134.7, 130.6,127.3, 126.4, 126.0, 125.7, 119.3, 116.4, 108.4, 98.7, 71.9, 70.8, 70.6,70.5, 68.8, 65.4, 59.0, 49.4, 25.5, 24.3; HRMS Calc for C₂₆H₃₂N₂O₅(M+H)⁺453.2384 found 453.2390.

Example 1g (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-cyanoacetate (12)

To a solution of 2-cyanoacetic acid (1.02 g, 12 mmol), the acetal(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (1.32 g, 10 mmol) in 5 ml ofDCM and DMAP (61 mg, 0.50 mmol) was added dropwise at 0° C. Finally, EDC1.86 g (12 mmol) was added and the reaction mixture was stirred at 0° C.for 6 hours. The reaction was diluted with 15 mL of DCM and the formedDCU was filtered off. The filtrate was dried over anhydrous MgSO₄ andthe solvents were removed under reduced pressure. The residue waspurified by flash chromatography (Hex: EtOAc; 10:1) to give compound 12.12: 71% yield; colorless liquid; ¹H NMR (400 MHz, CDCl₃) δ 4.34-4.32 (m,1H), 4.28-4.17 (m, 2H), 4.07 (dd, 1H, J=6.5 Hz, J=8.5 Hz), 3.75 (dd, 1H,J=5.8 Hz, J=8.5 Hz), 3.51 (s, 2H), 1.41 (s, 3H), 1.34 (s, 3H); HRMS Calcfor C₉H₁₃NO₄(M+H)⁺ 200.0923 found 200.0931.

Example 1h(E)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl2-cyano-3-(4-(dimethylamino)phenyl)acrylate(13)

To a round bottom flask containing a solution of aldehyde 6a (0.75 g,5.0 mmol) and compound 12 (1.2 g, 5.5 mmol) in 20 ml of THF was added0.50 mmol of piperidine and the mixture was heated at 50° C. The crudemixture was concentrated under reduced pressure and the product waspurified via flash chromatography (10-30% ethyl acetate in hexane) togive compound 13. 13: 91% yield; yellow solid; ¹HNMR (400 MHz, CDCl₃) δ8.08 (s, 1H), 7.94 (d, 2H, J=9.0 Hz), 6.69 (d, 2H, J=9.2 Hz), 4.42-4.29(m, 3H), 4.13 (dd, 1H, J=6.2 Hz, J=8.6 Hz), 3.89 (dd, 1H, J=5.9 Hz,J=8.5 Hz), 3.11 (s, 6H), 1.46 (s, 3H), 1.38 (s, 3H); ¹³CNMR (400 MHz,CDCl₃) δ 164.3, 155.3, 153.9, 134.5, 119.5, 117.5, 111.7, 110.1, 93.3,73.7, 66.7, 65.6, 40.3, 26.9, 25.7; HRMS Calc for C₁₈H₂₂N₂O₄(M+H)⁺331.1658 found 331.1691.

Example 1i (E)-2,3-dihydroxypropyl 2-cyano-3-(4-(dimethylamino)phenyl)acrylate (14)

Compound 13 (0.5 g, 1.5 mmol) was dissolved in a mixture of THF/MeOH(1:1) and DOWEX-H⁺ resin (0.10 g) was added and the heterogeneousmixture was stirred for 20 hours. The DOWEX-H⁺ resin was removed byfiltration and triethylamine (50 mg, 0.5 mmol) was added and the solventwas removed under reduced pressure. The residue was purified by flashchromatography (100% ether) to give compound 14. 14: 75% yield; brightyellow solid; ¹H NMR (400 MHz, CDCl₃) δ 8.08 (s, 1H), 7.94 (d, 2H, J=9.1Hz), 6.69 (d, 2H, J=9.2 Hz), 4.42-4.32 (m, 2H), 4.05 (m, 1H), 3.80-3.70(m, 2H), 3.12 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 199.0, 164.8, 155.5,154.0, 134.6, 119.4, 117.9, 111.8, 111.7, 92.8, 70.3, 70.2, 66.9, 66.8,63.6, 63.5, 40.3, 40.2; HRMS Calc for C₁₅H₁₈N₂O₄ (M+H)⁺ 291.1345 Found291.1361.

Example 1j Diethyl 4-bromobenzylphosphonate (16)

1-bromo-4-(bromomethyl) benzene (5.0 g, 20 mmol) and triethyl phosphite(51 mL, 300 mmol) were mixed in a round bottom flask and refluxed at 90°C. for 19 hours. Excess triethyl phosphite was removed under reducedpressure and the product purified by flash chromatography (1:1Hexane/EtOAc) to give compound 16. 16: 98% yield; colorless liquid; ¹HNMR (400 MHz, CDCl₃) δ 7.30 (d, 2H, J=7.5 Hz), 7.05 (d, 2H, J=7.6 Hz),3.99-3.88 (m, 4H), 2.99 (s, 1H), 2.94 (s, 1H), 1.12 (t, 6H, J=6.9 Hz);¹³C NMR (100 MHz, CDCl₃) δ 131.7, 131.6, 131.5, 121.0, 62.3, 34.0, 32.0,16.5; HRMS Calc for C₁₁H₁₆BrO₃P (M+H)⁺ 307.0097 found 307.0093.

Example 1k (E)-4-(4-bromostyryl)-N,N-dimethylaniline (17)

DMF (anhydrous) (10.5 mL) was added to sodium methoxide (176 mg, 3.26mmol) and the color was changed to pink. To the above solution diethyl4-bromobenzylphosphonate (1.0 g, 3.26 mmol) in DMF (6.5 ml) was addeddropwise over 2 minutes, followed by 4 (dimethylamino)benzaldehyde (486mg, 3.26 mmol). The reaction mixture was stirred at room temperature for24 hours. Deionized water (17 mL) was added. The product was filteredout through vacuum filtration and recrystallized with DCM/hexane to givecompound 17. 17: 74%. Yield; tan solid; ¹H NMR (400 MHz, CDCl₃) δ7.47-7.32 (m, 6H), 7.04 (d, 1H, J=12.5 Hz), 6.83 (d, 1H, J=16.3 Hz),6.71 (d, 2H, J=8.9 Hz), 2.99 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 150.5,137.4, 136.1, 132.1, 131.8, 129.7, 128.3, 128.2, 127.9, 127.7, 125.5,123.2, 120.3, 112.6, 40.7; HRMS Calc for C₁₆H₁₆BrN 302.0541 found302.0539.

Example 1l 4-(4-(dimethylamino)styryl)benzaldehyde (18)

To a round bottom flask compound 17 (300 mg, 1 mmol) was transferredfollowed by THF (5 mL). The heterogeneous solution was cooled at −78° C.and n-BuLi (1.6M in hexane, 1 mmol) was added dropwise over 5 min,followed by DMF (1.5 mL). The reaction mixture was stirred at −78° C.for 3 hours then it was quenched by water (1 mL) and the mixture wasextracted with ether (2×25 mL). The combined organic extracts werewashed with brine, dried over MgSO₄ and concentrated under reducedpressure to give compound 18. 18: 60% yield; yellow powder; ¹H NMR (400MHz, CDCl₃) δ 9.96 (s, 1H), 7.83 (d, 2H, J=8.2 Hz), 7.60 (d, 2H, J=8.2Hz), 7.44 (d, 2H, J=8.8 Hz), 7.22 (d, 1H, J=16.2 Hz), 6.94 (d, 1H,J=16.2 Hz), 6.72 (d, 2H, J=8.8 Hz), 3.01 (s, 6H); ¹³C NMR (100 MHz,CDCl₃) δ 191.8, 150.8, 144.7, 134.7, 134.6, 132.7, 130.4, 128.4, 126.4,124.9, 122.8, 112.4, 40.5; HRMS calc for C₁₇H₁₇NO 252.1384 found252.1383.

Example 1m(E)-2-(2-(2-methoxyethoxyl)ethoxy)ethyl-cyano-3-(4-(4(dimethylamino)styryl)phenyl)acrylate(19)

97% yield; red solid; ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 7.98 (d,2H, J=8.4 Hz), 7.57 (d, 2H, J=8.4 Hz), 7.45 (d, 2H, J=8.7 Hz), 7.20 (d,1H, J=16.2 Hz), 6.92 (d, 1H, J=16.2 Hz), 6.72 (d, 2H, J=8.7 Hz), 4.47(m, 2H), 3.84-3.82 (m, 2H), 3.74-3.72 (m, 2H), 3.70-3.66 (m, 4H),3.57-3.55 (m, 2H), 3.37 (s, 3H), 3.02 (s, 6H); ¹³C NMR (100 MHz, CDCl₃)δ 154.9, 133.0, 132.2, 128.6, 128.5, 126.7, 122.7, 112.4, 72.2, 71.1,70.8, 69.0, 65.8, 59.3, 40.6, 40.5, 29.9, 28.2; HRMS calc forC₂₇H₃₂N₂O₅(M+Na)⁺ 487.2203 found 487.2201.

Example 2 Compound Synthesis of Compounds 27-33

Results of analysis of compounds described herein are provided inExamples 2a-2o following.

General Notes.

All reagents were purchased at highest commercial quality and usedwithout further purification except where noted. Air- andmoisture-sensitive liquids and solutions were transferred via syringe orstainless steel cannula. Organic solutions were concentrated by rotaryevaporation below 45° C. at approximately 20 mmHg. All non-aqueousreactions were carried out under anhydrous conditions. Yields refer tochromatographically and spectroscopically (¹H NMR, ¹³C NMR) homogeneousmaterials, unless otherwise stated. Reactions were monitored bythin-layer chromatography (TLC) carried out on 0.25 mm DynamicAdsorbents, Inc. silica gel plates (60F-254) and visualized under UVlight and/or developed by dipping in solutions of 10% ethanolicphosphomolybdic acid (PMA) and applying heat. Dynamic Adsorbents, Inc.silica gel (60, particle size 0.040-0.063 mm) was used for flashchromatography. NMR spectra were recorded on the Varian Mercury 400, 300and/or Unity 500 MHz instruments and calibrated using the residualnon-deuterated solvent as an internal reference. The followingabbreviations were used to explain the multiplicities: s=singlet,d=doublet, t=triplet, q=quartet, m=multiplet, b=broad. High resolutionmass spectra (HRMS) were recorded on a VG 7070 HS mass spectrometerunder electron spray ionization (ESI) or electron impact (EI)conditions.

A general strategy for the synthesis of compounds described hereinincluding compounds 27-33 is depicted in scheme 5. Commerciallyavailable methyl 6-bromo naphthalene-2-carboxylate (20) was converted tothe corresponded naphthaldehyde 21 in two steps: a) reduction of theester to the primary alcohol by using DIBALH and b) oxidation of thealcohol to the aldehyde after treatment with PCC. Granzhan, A.;Teulade-Fichou, M.-P., Tetrahedron 2009, 65, (7), 1349-1360. Thetransformation of the bromide to the appropriate amine demanded the useof novel chemistry to improve the yield and apply the method in biggerscale. Treatment of bromide 21 in the presence of palladium usingBuchwald and Hartwig conditions resulted aldehydes 22-25 in excellentyield for most cases. Guram, A. S.; Rennels, R. A.; Buchwald, S. L.,Angew. Chem. Int. Ed. Engl. 1995, 34, (12), 1348-1350; Wolfe, J. P.;Buchwald, S. L., J. Org. Chem. 2000, 65, (4), 1144-1157; Hartwig, J. F.,Accounts Chem. Res. 2008, 41, (11), 1534-1544. Knovenagel condensationof aldehydes 22-25 and the appropriate cyanoester 26 concluded thesynthesis of the final probes 27-32. Sutharsan, J.; Lichlyter, D.;Wright, N. E.; Dakanali, M.; Haidekker, M. A.; Theodorakis, E. A.,Tetrahedron 2010, 66, (14), 2582-2588. Deprotection of the acetal inprobe 32 by using acidic resin, yielded the final dye 33. Table 3summarizes the R, X combinations of the final products and thecondensation yields.

TABLE 3 Structures and yields for the Aβ binding probes Compound X RYield 27

90% 28

85% 29

83% 30

87% 31

89% 32

83% 33

84%

Example 2a 6-bromo-2-naphtaldehyde (21)

To a solution of DIBAL-H (1.0 M in heptane, 34 mL, 34 mmol) at 0° C.under argon, a solution of 20 (3.0 gr, 11 mmol) in anhydrous THF wasadded dropwise. The reaction mixture was allowed to warm up to roomtemperature and left stirring overnight. Upon completion, MeOH wasadded, followed by a saturated sodium potassium tartrate solution andethylacetate. After the two phases were separated, the organic phase waswashed with saturated solution of ammonium chloride and brine, driedover MgSO₄ and concentrated under reduced pressure to yield6-bromo-2-(hydroxymethyl)naphthalene. ¹H NMR (400 MHz, CDCl₃): δ 7.99(bs, 1H), 7.77 (bs, 1H), 7.74 (d, 1H, J=8.5 Hz), 7.69 (d, 1H, J=8.7 Hz),7.55 (dd, 1H, J=1.7 Hz, J=8.7 Hz), 7.49 (dd, 1H, J=1.7 Hz, J=8.5 Hz),4.84 (bs, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 138.8, 133.9, 131.7, 129.7,129.5, 129.5, 127.4, 126.1, 125.2, 119.8, 65.2.

To a suspension of pyridinium chlorochromate (2.4 gr, 11 mmol) inanhydrous CH₂Cl₂ (60 mL) was added a solution of the above alcohol inanh. CH₂Cl₂ and the reaction was heated under reflux for 5 hours. Uponcompletion, it was cooled to room temperature and poured into diethylether. The solution was then filtered through a pad of silica andconcentrated under reduced pressure to yield 21 (2.4 gr, 95%). 20: whitesolid; ¹H NMR (400 MHz, CDCl₃) δ 10.15 (s, 1H), 8.31 (bs, 1H), 8.08 (bs,1H), 7.98 (dd, 1H, J=1.5 Hz, J=8.5 Hz), 7.86 (m, 2H), 7.67 (dd, 1H,J=1.5 Hz, J=8.5 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 191.8, 137.3, 134.3,134.1, 131.0, 131.0, 130.6, 130.2, 128.2, 124.0, 123.

General Procedure for the Synthesis of 6-amino-substitutedNaphtaldehydes (Cmpds 22-25)

In dry and degassed toluene (0.8 mL), were added Pd(OAc)₂ (0.022 mmol)and P(tBu)₃ (0.078 mmol) After stirring for 20 min, 8 (0.207 mmol), theappropriate amine (0.249 mmol) and Cs₂CO₃ (0.280 mmol) were added andthe reaction left stirring for three days under reflux. After threedays, the reaction was cooled at room temperature, diluted with CH₂Cl₂,filtered, concentrated under reduced pressure and purified via silicagel flash chromatography (hexanes/EtOAc 0-10%).

Example 2b 6-(piperidin-1-yl)naphthalene-2-carbaldehyde (22)

70% yield, yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 10.03 (s, 1H), 8.15(s, 1H), 7.83 (m, 2H), 7.68 (d, 1H, J=8.6 Hz) 7.32 (dd, 1H, J=2.4 Hz,J=9.1 Hz), 7.08 (d, 1H, J=2.4 Hz), 3.38 (m, 4H), 1.78-1.63 (m, 6H); ¹³CNMR (100 MHz, CDCl₃) δ 191.9, 151.9, 138.5, 134.4, 131.3, 130.4, 127.2,126.3, 123.4, 119.5, 108.8, 49.6, 25.5, 24.3; HRMS Calc for C₁₆H₁₈NO(M+H)⁺ 240.1383 found 240.1381.

Example 2c 6-morpholinonaphthalene-2-carbaldehyde (23)

79% yield, yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 10.06 (s, 1H), 8.20(s, 1H), 7.88 (m, 2H), 7.73 (d, 1H, J=8.4 Hz), 7.32 (m, 1H), 7.11 (d,1H, J=1.2 Hz), 3.92 (m, 4H), 3.36 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ191.9, 151.3, 138.1, 134.2, 131.8, 130.6, 127.5, 127.0, 123.6, 118.7,109.0, 66.7, 48.5; HRMS Calc for C₁₅H₁₅NO₂Na (M+Na)⁺ 264.0995 found264.0996.

Example 2d 6-(4-methylpiperazin-1-yl)naphthalene-2-carbaldehyde (24)

77% yield, yellow solid; ¹H NMR (300 MHz, CDCl₃) δ 10.00 (s, 1H), 8.13(s, 1H), 7.80 (m, 2H), 7.66 (d, 1H, J=8.6 Hz), 7.28 (dd, 1H, J=2.1 Hz,J=9.2 Hz), 7.06 (d, 1H, J=2.1 Hz), 3.36 (m, 4H), 2.57 (m, 4H), 2.33 (s,3H); ¹³C NMR (100 MHz, CDCl₃) δ 191.5, 151.0, 138.0, 134.0, 131.3,130.2, 127.1, 126.4, 123.1, 118.7, 108.7, 54.5, 47.8, 45.7; HRMS Calcfor C₁₆H₁₉N₂O (M+H)⁺ 255.1492 found 255.1491.

Example 2e 6-(2-morpholinoethylamino)naphthalene-2-carbaldehyde (25)

33% yield, yellow solid; ¹H NMR (400 MHz, CDCl₃) δ 10.01 (s, 1H), 8.14(s, 1H), 7.83 (dd, 1H, J=1.6 Hz, J=8.6 Hz), 7.76 (d, 1H, J=8.9 Hz), 7.64(d, 1H, J=8.6 Hz), 6.98 (dd, 1H, J=2.3 Hz, J=8.9 Hz), 6.79 (d, 1H, J=2.3Hz), 4.88 (bs, 1H), 3.75 (m, 4H), 3.31 (dd, 2H, J=5.1 Hz, J=11.1 Hz),2.72 (m, 2H), 2.51 (bs, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 191.9, 148.8,139.1, 134.6, 130.8, 130.7, 126.6, 126.0, 123.8, 118.7, 103.8, 66.9,56.7, 53.3, 39.3; HRMS Calc for C₁₇H₂₁N₂O₂ (M+H)⁺ 285.1598 found285.1600.

General Procedure for the Synthesis of 2-cyanoacetates (26)

To a solution of 2-cyanoacetic acid (2.72 mmol), the appropriate alcohol(2.27 mmol) in CH₂Cl₂ (2.5 mL) and DMAP (0.013 mmol) was added dropwiseat 0° C. Finally, DCC (2.72 mmol) was added and the reaction mixture wasstirred at 0° C. for 6 hours. The reaction was diluted with CH₂Cl₂ andthe formed DCU was filtered off. The filtrate was dried over MgSO₄ andconcentrated under reduced pressure. The residue was purified by silicagel flash chromatography to yield 2-cyanoacetate 7.

Example 2f 2-(2-(2-methoxyethoxyl)ethoxy)ethyl-2-cyanoacetate (26a)

86% yield; colorless liquid; ¹H NMR (400 MHz, CDCl₃) δ 4.29 (m, 2H),3.67, (m, 2H), 3.59 (m, 6H), 3.50 (m, 2H), 3.49 (s, 2H), 3.32 (s, 3H);¹³C NMR (100 MHz, CDCl₃) δ 163.0, 113.0, 71.7, 70.4, 70.3, 68.3, 65.5,58.8, 24.5; HRMS: calcd. for C₁₀H₁₇NO₅: (M+H⁺) 232.1185. found 232.1199.

Example 2g 2-(2-(2-(2-methoxyethoxyl)ethoxy)ethoxy)ethyl-2-cyanoacetate(26b)

68% yield; colorless liquid; ¹H NMR (400 MHz, CDCl₃) δ 4.21 (bs, 2H),3.61 (bs, 2H), 3.51 (m, 12H), 3.43 (m, 2H), 3.24 (bs, 3H) ¹³C NMR (100MHz, CDCl₃) δ 163.0, 113.0, 71.4, 70.2, 70.1, 70.1, 70.0, 68.1, 65.3,58.5, 24.2;

Example 2h (2,2-dimethyl-1,3-dioxolan-4-yl)methyl 2-cyanoacetate (26c)

71% yield; colorless liquid; ¹H NMR (400 MHz, CDCl₃) δ 4.35 (m, 1H),4.29-4.19 (m, 2H), 4.09 (m, 1H), 3.76 (dd, 1H, J=5.8 Hz, J=8.6 Hz), 3.52(s, 2H), 1.43 (s, 3H), 1.36 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 162.8,112.7, 110.1, 73.0, 66.8, 65.9, 26.6, 25.2, 24.6; HRMS Calc for C₉H₁₃NO₄(M+H)⁺ 200.0923 found 200.0931.

General Procedure for the Synthesis of Fluorescent Probes 27-33

To a round bottom flask containing a solution of aldehyde (0.21 mmol)and the appropriate 2-cyanoacetate (0.23 mmol) in THF (0.8 mL),piperidine (0.02 mmol) was added and the mixture left stirring at 50° C.The reaction was monitored by TLC and was completed within 21 hours. Thecrude mixture was concentrated under reduced pressure and the productwas purified via flash column chromatography (10-30% EtOAc in hexanes).

Example 2i (E)-2-(2-(2-methoxyethoxyl)ethoxy)ethyl2-cyano-3-(2-(piperidin-1-yl)napthalen-6-yl)acrylate (27)

90% yield; red liquid; ¹H NMR (400 MHz, CDCl₃) δ 8.31 (s, 1H), 8.22 (bs,1H), 8.10 (d, 1H, J=8.8 Hz), 7.76 (d, 1H, J=9.2 Hz), 7.65 (d, 1H, J=8.8Hz), 7.30 (dd, 1H, J=2.1 Hz, J=9.2 Hz), 7.05 (d, 1H, J=2.1 Hz), 4.47 (m,2H), 3.83 (m, 2H), 3.74-3.66 (m, 6H), 3.56 (m, 2H), 3.42-3.38 (m, 4H),3.37 (s, 3H), 1.74 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 163.3, 155.4,151.9, 137.7, 134.7, 130.6, 127.2, 126.4, 125.9, 125.6, 119.2, 116.4,108.3, 71.8, 70.7, 70.5, 70.5, 68.7, 65.3, 58.9, 49.3, 25.4, 24.3; HRMSCalc for C₂₆H₃₂N₂O₅Na (M+Na)⁺ 475.2203 found 475.2197.

Example 2j (E)-2-(2-(2-methoxyethoxyl)ethoxy)ethyl2-cyano-3-(2-(4-methylpiperazin-1-yl)napthalen-6-yl) acrylate (28)

85% yield; red liquid; ¹H NMR (400 MHz, CDCl₃) δ 8.31 (s, 1H), 8.23 (s,1H), 8.10 (d, 1H, J=8.6 Hz), 7.78 (d, 1H, J=9.1 Hz), 7.67 (d, 1H, J=8.6Hz), 7.29 (d, 1H, J=9.1 Hz), 7.06 (s, 1H), 4.46 (m, 2H), 3.83 (m, 2H),3.73 (m, 2H), 3.67 (m, 4H), 3.55 (m, 2H) 3.42 (bs, 4H), 3.36 (s, 3H),2.61 (bs, 4H), 2.37 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 163.2, 155.4,151.4, 137.5, 134.5, 130.6, 127.4, 126.9, 126.1, 126.1, 119.0, 116.2,108.7, 99.3, 71.9, 70.8, 70.6, 70.5, 68.8, 65.4, 59.0, 54.8, 48.0, 46.1;HRMS Calc for C₂₆H₃₄N₃O₅ (M+H)⁺ 468.2493 found 468.2494.

Example 2k (E)-2-(2-(2-methoxyethoxyl)ethoxy)ethyl2-cyano-3-(2-morpholinonapthalen-6-yl)acrylate (29)

83% yield; red liquid; ¹H NMR (300 MHz, CDCl₃) δ 8.31 (s, 1H), 8.24 (s,1H), 8.11 (dd, 1H, J=1.9 Hz, J=8.8 Hz), 7.80 (d, 1H, J=9.1 Hz), 7.69 (d,1H, J=8.8 Hz), 7.28 (m, 1H), 7.06 (d, 1H, J=1.9 Hz), 4.47 (m, 2H), 3.90(m, 4H), 3.83 (m, 2H), 3.70 (m, 6H), 3.55 (m, 2H), 3.35 (m, 7H); ¹³C NMR(100 MHz, CDCl₃) δ 163.0, 155.2, 151.3, 137.2, 134.4, 130.6, 127.4,127.0, 126.1, 126.0, 118.5, 116.1, 108.5, 99.4, 71.8, 70.7, 70.5, 70.4,68.6, 66.5, 65.3, 58.9, 48.2; HRMS Calc for C₂₅H₃₀N₂O₆Na (M+Na)⁺477.1996 found 477.1995.

Example 2l (E)-2-(2-(2-methoxyethoxyl)ethoxy)ethyl3-(2-(2-morpholinoethylamino) naphthalen-6-yl)-2-cyano acrylate (30)

87% yield; red liquid; ¹H NMR (500 MHz, CDCl₃) δ 8.28 (s, 1H), 8.19 (d,1H, J=1.6 Hz), 8.08 (dd, 1H, J=1.9 Hz, J=8.8 Hz), 7.69 (d, 1H, J=8.9Hz), 7.60 (d, 1H, J=8.8 Hz), 6.95 (dd, 1H, J=2.3 Hz, J=8.8 Hz), 6.74 (d,1H, J=2.2 Hz), 4.96 (bs, 1H), 4.46 (m, 2H), 3.83 (m, 2H), 3.76-3.72 (m,6H), 3.69-3.65 (m, 4H), 3.57-3.54 (m, 2H), 3.36 (s, 3H), 3.31 (s, 2H),2.70 (m, 2H), 2.51 (s, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 163.4, 155.5,149.0, 138.3, 135.0, 130.9, 126.6, 126.3, 126.2, 124.9, 118.9, 116.5,103.6, 98.2, 71.9, 70.8, 70.6, 70.5, 68.8, 66.9, 65.3, 59.0, 56.6, 53.2,39.2; HRMS Calc for C₂₇H₃₆N₃O₆ (M+H)⁺ 498.2599 found 498.2596.

Example 2lm (E)-2-(2-(2-(2-methoxyethoxyl)ethoxy)ethoxy)ethyl2-cyano-3-(2-(piperidin-1-yl)napthalen-6-yl) acrylate (31)

89% yield; red liquid; ¹H NMR (400 MHz, CDCl₃) δ 8.20 (s, 1H), 8.10 (s,1H), 8.01 (d, 1H, J=8.5 Hz), 7.66 (d, 1H, J=9.0 Hz), 7.55 (d, 1H, J=9.0Hz), 7.20 (d, 1H, J=8.5 Hz), 6.95 (s, 1H), 4.39 (bs, 2H), 3.75 (bs, 2H),3.65-3.54 (m, 10H), 3.45 (m, 2H), 3.31 (bs, 4H), 3.28 (s, 3H), 1.65-1.54(m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 163.2, 155.3, 151.8, 137.6, 134.6,130.5, 127.1, 126.3, 125.8, 125.5, 119.1, 116.3, 108.2, 98.4, 71.7,70.6, 70.4, 70.3, 68.6, 65.3, 58.8, 49.2, 25.3, 24.2; HRMS Calc forC₂₈H₃₆N₂O₆Na (M+Na)⁺ 519.2466 found 519.2468.

Example 2n (E)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl2-cyano-3-(2-(piperidin-1-yl)naphthalene-6-yl) acrylate (32)

83% yield; red liquid; ¹H NMR (400 MHz, CDCl₃) δ 8.29 (s, 1H), 8.19 (s,1H), 8.09 (dd, 1H, J=1.9 Hz, J=8.8 Hz), 7.74 (d, 1H, J=9.3 Hz), 7.63 (d,1H, J=8.8 Hz), 7.28 (dd, 1H, J=2.8 Hz, J=9.3 Hz), 7.03 (d, 1H, J=1.9Hz), 4.43 (m, 1H), 4.36 (m, 2H), 4.14 (dd, 1H, J=6.0 Hz, J=8.5 Hz), 3.90(dd, 1H, J=6.0 Hz, J=8.5 Hz), 3.40 (m, 4H), 1.73-1.66 (m, 6H), 1.48 (s,3H), 1.39 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 162.9, 155.4, 151.5,137.6, 134.6, 130.5, 127.0, 126.2, 125.6, 125.3, 119.0, 116.1, 109.6,108.2, 98.0, 73.1, 65.9, 65.6, 57.1, 49.1, 45.5, 29.4, 26.4, 25.2, 24.0;MS (M+H)⁺ 421.24.

Example 2o (E)-2,3-dihydroxypropyl2-cyano-3-(2-(piperidin-1-yl)naphthalene-6-yl)acrylate (33)

Compound 31 (50 mgr, 0.12 mmol) was dissolved in a mixture of THF/MeOH(1:1) and DOWEX-H⁺ resin (15 mgr) was added and the heterogeneousmixture was stirred for 20 hours. The resin was removed by filtrationand triethylamine was added and the solvent was removed under reducedpressure. The residue was purified by flash chromatography to givecompound 32. 32: 38 mgr, 84% yield; red liquid; ¹H NMR (400 MHz, CDCl₃)δ 8.30 (s, 1H), 8.19 (s, 1H), 8.08 (d, 1H, J=8.8 Hz), 7.74 (d, 1H, J=9.1Hz), 7.63 (d, 1H, J=9.1 Hz), 7.29 (m, 1H), 7.03 (s, 1H), 4.46-4.36 (m,2H), 4.09 (m, 1H), 3.81 (dd, 1H, J=5.5 Hz, J=11.3 Hz), 3.73 (dd, 1H,J=5.5 Hz, J=11.3 Hz), 3.41 (m, 4H), 1.74-1.67 (m, 6H); ¹³C NMR (100 MHz,CDCl₃) δ 163.6, 156.0, 152.0, 137.9, 135.0, 130.7, 127.3, 126.3, 125.9,125.5, 119.2, 116.6, 108.3, 97.8, 69.9, 67.0, 63.2, 49.3, 25.5, 24.3;HRMS Calc for C₂₂H₂₅N₂O₄ (M+H)⁺ 381.1809 found 381.1802.

Example 3 Detection and Binding Studies

Studies for Compounds 8a-8d, 11, 14 and 18.

An initial study to determine whether a compound can associate withaggregated Aβ is to compare its fluorescence spectra before and aftermixing with the Aβ aggregates. See E. E. Nesterov, J. Skoch, B. T.Hyman, W. E. Klunk, B. J. Bacskai, T. M. Swager, Angew. Chem. Int. Edit.2005, 44:5452-5456; Z. P. Zhuang, M. P. Kung, H. F. Kung, J. Med. Chem.2006, 49:2841-2844; Q. A. Li, J. S. Lee, C. Ha, C. B. Park, G. Yang, W.B. Gan, Y. T. Chang, Angew. Chem. Int. Edit. 2004, 43:6331-6335; H. F.Kung, C. W. Lee, Z. P. Zhuang, M. P. Kung, C. Hou, K. Plossl, J. Am.Chem. Soc. 2001, 123:12740-12741. Typically, a fluorescentamyloid-binding agent displays a significant fluorescence intensityincrease after binding to Aβ aggregates as compared to its nativefluorescence in solution. See H. LeVine III, Protein Sci. 1993,2:404-410. Along these lines we measured the fluorescent properties ofeach compound at 4 μM concentration before and after mixing withpreaggregated Aβ(1-42) peptides (5 μM, aggregated in PBS buffer for 3days at 25° C.).

In all cases, a 1.3-9.4 fold fluorescence intensity increase wasobserved in the presence of aggregated Aβ, indicating that thesecompounds bind to the peptide (Table 2). In most cases a modestblue-shift (6-20 nm) was observed upon binding. Only in the case of thenaphthalene-based Cmpd 11 was a significant red shift of 76 nm observedupon binding to preaggregated Aβ (FIGS. 1 c, 1 d). Interestingly, thisbinding was accompanied with a 9.3 fold intensity increase. A similarintensity increase has been observed with FDDNP and may be explained bythe ability of the naphthalene motif to create excimers upon binding toits target. See E. D. Agdeppa, V. Kepe, J. Liu, S. Flores-Torres, N.Satyamurthy, A. Petric, G. M. Cole, G. W. Small, S. C. Huang, J. R.Barrio, J. Neurosci. 2001, 21:1-5; S. Abad, I. Vaya, M. C. Jimenez, U.Pischel, M. A. Miranda, ChemPhysChem 2006, 7:2175-2183; C. Spies, R.Gehrke J. Phys. Chem. A 2002, 106:5348-5352.

Cmpds 8a and 8b exhibited similar fluorescence characteristicssuggesting that addition of a methoxy group on the phenyl group does notalter the binding properties of the compound as a probe. On the otherhand, it is worth noting that increasing the size of the alkyl groups ofthe nitrogen leads to a significant increase in the fluorescenceintensity after binding (Table 4, 8a, 8c, 8d). This is likely a resultof the decreased rotational freedom of the molecules upon binding to theaggregated forms of Aβ peptide. See W. Schuddeboom, S. A. Jonker, J. M.Warman, U. Leinhos, W. Kuehnle, K. A. Zachariasse, J. Phys. Chem. 1992,96:10809-10819; Y. V. Il'chev, W. Kuehnle, K. A. Zachariasse, J. Phys.Chem. 1998, 102:5670-5680. Interestingly, no increase of fluorescenceintensity was observed upon mixing of these compounds with monomeric Aβpeptide. This supports the notion that these compounds bind selectivelyto aggregated forms of Aβ. The fluorescence profile of 8d (excitationand emission) is shown in FIG. 1A and FIG. 1B.

TABLE 4 Fluorescence profile, Kd, IC₅₀ and related values for theinteraction of the synthesized compounds with aggregated Aβ(1-42)peptides Excitation Excitation Emission Emission Maximum max before maxafter max before max after % IC₅₀ Comp binding binding binding bindingFold K_(d) inhibition (μM) No (nm) (nm) (nm) (nm) increase (μM) R²(ELISA) (ELISA) LogP 8a 439 435 476 470 1.8 2.6 0.93 81 129.0 1.74 8b442 444 478 469 1.3 5.3 0.99 92 1.2 1.54 8c 445 442 478 470 4.2 4.8 0.9698 11.4 2.49 8d 432 440 466 468 9.4 4.4 0.95 91 90.6 4.62 11  445 440462 538 9.3 2.5 0.98 58 74.3 3.81 14  437 434 476 467 2.2 3.3 0.99 7982.1 1.07 19  312 319 658 638 2.3 1.4 0.98 40 33.6 4.30

Aggregated Aβ peptide was prepared by dissolving Aβ(1-42) in PBS pH 7.4to a final concentration of 100 μM. This solution was magneticallystirred at 1200 rpm for 3 days at room temperature. The 100 μM Aβ(1-42)stock solution in PBS was aliquoted and frozen at 80° C. for up to 4weeks without noticeable change in its property. 150 μL ofpre-aggregated Aβ(1-42) was added to 2.85 mL of compound to attain afinal concentration of 5 μM Aβ(1-42) and 4 μM of compound. The solutionwas transferred to 3 mL cuvette and the fluorescence measured at 25° C.As shown in FIG. 4, association of compounds described herein withaggregated Aβ provides changes in both excitation and emission spectra.The fluorescence excitation spectra of Cmpds 8a, 8b, 8c, 14 and 19 aredepicted in FIG. 4, respectively.

We also measured the apparent binding constants (Kd) of the compounds(in concentrations of 10, 5, 2.5 and 1.25 μM) to 5.0 μM pre-aggregatedAβ(1-42) peptide. The Kd can be measured from the double reciprocal ofthe fluorescent maximum and the concentration of the compound. See H.LeVine III, Protein Sci. 1993, 2:404-410. All Kd values were measuredbetween 1.4 and 5.3 μM (Table 4). It is remarkable that, despite thestructural differences, these compounds display similar Kd valuessuggesting that they bind in a similar fashion to aggregated Aβ.Moreover, these values are similar to the reported Kd values for ThT (2μM). [22,] See LeVine, Id.; Lockhart, L. Ye, D. B. Judd, A. T. Merritt,P. N. Lowe, J. L. Morgenstern, G. Z. Hong, A. D. Gee, J. Brown, J. Biol.Chem. 2005, 280:7677-7684; M. Biancalana, K. Makabe, A. Koide, S. Koide,J. Mol. Biol. 2008, 383:205-213; M. Biancalana, K. Makabe, A. Koide, S.Koide, J. Mol. Biol. 2009, 385:1052-1063. The double reciprocal plot offluorescence intensity versus concentration of Cmpds 8d and 11 are shownin FIG. 2. The Kd corresponds to the −1/(x-intercept) of the linearregression.

The association of the synthesized compounds with aggregated Aβ peptideswas tested using a semi-quantitative ELISA based assay developed by Yangand co-workers. See P. Inbar, J. Yang, Bioorg. Med. Chem. Lett. 2006,16:1076-1079; P. Inbar, C. Q. Li, S. A. Takayama, M. R. Bautista, J.Yang, ChemBioChem 2006, 7:1563-1566; P. Inbar, M. R. Bautista, S. A.Takayama, J. Yang, Anal. Chem. 2008, 80:3502-3506. The assay is based onscreening for molecules that inhibit the interaction of the aggregatedAβ peptide with a monoclonal anti-Aβ IgG raised against residues 1-17 ofAβ. Table 4 provides the concentrations of the compounds correspondingto 50% inhibition (IC₅₀) of the IgG-Aβ interactions as well as themaximal percentage of the IgG's inhibited from binding to the fibrils.All compounds exhibited IC₅₀ values at μM levels, the lowest value beingmeasured for 8b (IC50=1.17 μM). The maximum inhibition, a measure of theextent of surface coating of the aggregated peptide by the compounds,was determined to be between 40-98% (Table 4). See P. Inbar, J. Yang,Bioorg. Med. Chem. Lett. 2006, 16:1076-1079; P. Inbar, C. Q. Li, S. A.Takayama, M. R. Bautista, J. Yang, ChemBioChem 2006, 7:1563-1566; P.Inbar, M. R. Bautista, S. A. Takayama, J. Yang, Anal. Chem. 2008,80:3502-3506. Comparison of these data indicates that the surfacecoating increases by decreasing the size of the compound or the extentof the IC system. Specifically, while the maximum inhibition is between81-98% for the phenyl compounds, it decreases to 58% for the longernaphthalene compound 11 and to 40% for the more conjugated stilbene 19.Representative graphs for Cmpds 8d and 11 are shown in FIG. 3.Representative graphs for Cmpds 8a, 8b, 8c and 14 are shown in FIGS.7A-D, respectively.

The log P values for all the compounds were calculated to be between1.07 and 4.62 (Table 2) indicating that most of these compounds meet thesolubility criteria and should be able to cross the blood brain barrier.See P. Inbar, J. Yang, Bioorg. Med. Chem. Lett. 2006, 16:1076-1079; P.Inbar, C. Q. Li, S. A. Takayama, M. R. Bautista, J. Yang, ChemBioChem2006, 7:1563-1566; P. Inbar, M. R. Bautista, S. A. Takayama, J. Yang,Anal. Chem. 2008, 80:3502-3506; C. A. Lipinski, F. Lombardo, B. W.Dominy, P. J. Feeney, Adv. Drug Deliver. Rev. 1997, 23:3-25. Log Pvalues were calculated using the Molinspiration Chem-informaticssoftware.

Studies for Compounds 27-31 and 33.

Aggregated Aβ peptide was prepared by dissolving Aβ(1-42) in PBS pH 7.4to a final concentration of 100 μM. This solution was magneticallystirred at 1200 rpm for 3 days at room temperature. The 100 μM Aβ(1-42)stock solution in PBS was aliquoted and frozen at −10° C. for up to 4weeks without noticeable change in its property. 15 μL of thepre-aggregated Aβ(1-42) was added to 285 μL of the probe (5% DMSO innano-pure water) to attain a final concentration of 5 μM Aβ(1-42) and 4μM of the probe. The solution was transferred to a 300 mL cuvette andthe fluorescent measured. FIGS. 9A-F show fluorescence excitationspectra of Cmpds 27-31 and 33, respectively.

TABLE 5 Fluorescence profile, K_(d) and logP values of the synthesizedprobes with aggregated Aβ(1-42) peptides Exc. Em. max (nm) max (nm) FoldCmp. be- af- be- af- in- # fore ter fore ter crease K_(d) SD R² logP 27415 410 590 545 7.7 1.4 0.2 0.99 3.81 28 400 385 580 530 4.9 4.6 1.30.98 2.79 29 400 380 530 525 5.1 13.8 2.9 0.99 2.74 30 430 430 570 5402.9 6.7 2.1 0.98 2.53 31 420 410 590 546 8.3 1.6 0.3 0.93 3.60 33 410410 540 535 7.2 1.6 0.9 0.93 3.14

In principle, an amyloid-binding probe displays significant increase ofthe fluorescent emission upon binding with the aggregates as compared tothat in solution. LeVine III, H., Protein Sci. 1993, 2, (3), 404-410.Along these lines, we compared the fluorescent properties of 27-31 or 33in aqueous solution with or without the presence of aggregated Aβ42peptides. Specifically, we measured the fluorescent properties of eachdye at 4 μM concentration in nano pure water, before and after mixingwith aggregated Aβ42 peptide (final concentration peptide=5 μM). As itis shown in table 5, in all cases we observed a significant increase (3to 9-fold) in the intensity of the emission spectra of the probes uponassociation with the aggregated amyloid peptides. This intensityincrease was also accompanied with a blue shift in the emission spectraof around 5-50 nm. After binding, all compounds had excitation maximabetween 380-430 nm and their emission maxima were between 525-545 nm,suggesting that small changes in the donor or acceptor part of themolecule do not alter significantly their fluorescent maxima. However,compounds 27, 31 and 33, that possess piperidine as the electron donor,showed higher increase in fluorescence intensity after binding (7.7-,8.3- and 7.2-fold, respectively) compared to probes containingpiperazine, morpholine, or morpholino-ethanamine as electron donors.FIG. 9C provides a representative example of the fluorescent propertiesof compound 29. The figure shows fluorescent emission of compound 29before (solid line) and after (dotted line) mixing with Aβ aggregates.

We also measured the apparent binding constants (K_(d)) of the probes toaggregated Aβ42 peptides. The fluorescent intensity of each probe wasmeasured in concentrations of 1.25, 2.5, 5.0 and 10 μM in nano-purewater, mixed with 5 μM of the pre-aggregated Aβ42 peptides. Zhao, X.;Yang, J., ACS Chem. Neurosc. 1, (10), 655-660. In all cases the K_(d)values were at the μM level with compounds 27, 31 and 33 exhibiting thehighest affinity to aggregated Aβ peptides. The data from these bindingstudies suggests that small chemical modifications within thewater-solublizing region of the ANCA motif do not significantly affectthe binding of the probes to Aβ aggregates (compounds 27, 31 and 33). Onthe other hand, a decrease of the K_(d) value was observed uponchemically altering the electron donor of the ANCA scaffold. As shown intable 5, compounds having piperidine as the electron donor are found tohave lower K_(d) values (1.4-1.6 μM) compared to those possessingpiperazine, morpholine, or morpholino-ethanamine as electron donor(compounds 28, 29 and 30 respectively). FIGS. 10A-F show plots of thefluorescence intensity (at λ=525 nm) of compounds 27-31 and 32,respectively, as a function of the concentration in the presence ofaggregated Aβ42 peptides (5 μM) in solution. As an example, fitting thisdata for compound 29 revealed a K_(d) of 13.8 μM for association ofcompound 29 to aggregated Aβ42 peptides.

Finally, the lipophilicity (log P) of the synthesized probes wascalculated. All compounds were found to have log P values between 2.53and 3.81, suggesting that most of them fulfill the solubility criteriaand can potentially cross the blood brain barrier. Lipinski, C. A.;Lombardo, F.; Dominy, B. W.; Feeney, P. J., Adv. Drug Delivery Rev.1997, 23, (1-3), 3-25.

Example 6 Determination of Binding Constants for Compounds DescribedHerein

Pre-aggregated Aβ(1-42) (5 μM final concentration) was mixed withvarious concentrations of compounds described herein (10, 5, 2.5, 1.25μM) in PBS buffer (pH 7.4) and their fluorescence was measured. Thenegative inverse of the x-intercept of the linear regression, that wasdrawn between the double reciprocal of the fluorescence intensitymaximum and concentration of the compound, represents the compoundbinding constant (K_(d)) to Aβ(1-42).

Example 7 Determination of K_(d) from Fluorescence Method

In order to quantify the dissociation constants (K_(d)'s) for thebinding of fluorescent compounds with aggregated β-amyloid peptides, weused the method described by LeVine (see H. LeVine III, Protein Sci.1993, 2, 404-410). This method is similar to the method described byBenesi-Hildebran (see C. Yang, L. Liu, T. W. Mu, Q. X. Guo, Anal. Sci.2000, 16, 537-539). Here, the fluorescence of the compound was measuredwith and without the addition of the aggregated peptides in solution.The relative fluorescence enhancement of the compound upon binding toaggregated β-amyloid peptides was determined by taking the differencebetween F (fluorescence after the addition of aggregated peptides) and F(fluorescence before the addition of aggregated peptides).

In order to estimate the binding constant (K_(d)) for the compound-Aβcomplexes from the fluorescence studies, we made the followingassumptions:

-   -   1. All compounds are completely in solution and free of any        significant competing binding process such as self-aggregation.    -   2. The concentration of unbound compounds can be approximated as        close to the total concentration of the compounds.    -   3. The binding sites in the aggregated Aβ peptides are not        completely occupied at the concentration of Aβ-binding compounds        used for the fluorescence studies (i.e., the experiments are        carried out under non-saturated binding conditions).

According to the Beer-Lambert law (see J. W. Robinson, “Atomicspectroscopy”, 1996), one can obtain two expressions that relate theconcentration of bound compound ([HG]), free compound ([G]), and freeamyloid peptides ([H]) with either 1) the measured fluorescence of thecompound in solution before the addition of the aggregated peptides(F_(O)), or 2) the measured fluorescence of the compound in the presenceof the amyloid peptides (F):

F _(O)=ε_(G) l[G _(O)]  (1)

F=ε _(HG) l[HG]+ε _(H) l[H]+ε _(G) l[G]  (2)

where

[G_(O)]=total concentration of compound ε_(G)=absorption coefficient ofG

[G]=unbound compound concentration ε_(HG)=absorption coefficient of HG

[HG]=compound-Aβ complex concentration ε_(H)=absorption coefficient of H

[H_(O)]=total concentration of aggregated peptide l=path length

[H]=unbound aggregated peptide concentration.

Substituting [G_(O)]=[G]+[HG] into equation 1, and making theapproximation that ε_(HG)l[HG]+ε_(G)l[G]>>ε_(H)l[H], one can arrive at asimplified expression for the relative fluorescence of bound compound(ΔF):

ΔF=F−F _(O)=ε_(HG) l[HG]+ε _(G) l[G]−ε _(G) l[G]−ε _(G) l[HG]  (3)

or

ΔF=Δεl[HG]  (4)

where Δε=ε_(HG)−ε_(G).

In order to obtain a relationship between the change in measuredfluorescence of the compound (ΔF) with the binding constant of thecompound to aggregated β-amyloid peptides (K_(d)'s), we used thestandard equation for a binding isotherm to obtain a relationshipbetween [HG] and K_(d):

$\begin{matrix}{\lbrack{HG}\rbrack = \frac{\left\lbrack H_{O} \right\rbrack \lbrack G\rbrack}{K_{d} + \lbrack G\rbrack}} & (5)\end{matrix}$

Combining equation 4 and 5, we obtained a relationship between ΔF andK_(d):

$\begin{matrix}{{\Delta \; F} = {\frac{\left\lbrack H_{O} \right\rbrack \lbrack G\rbrack}{K_{d} + \lbrack G\rbrack}{\Delta\mathcal{E}}\; l}} & (6)\end{matrix}$

In order to estimate the K_(d) of the compound bound to aggregated Aβpeptides from the measured change in fluorescence, we take thereciprocal of the equation 6 to give:

$\begin{matrix}{\frac{1}{\Delta \; F} = {{\frac{K_{d}}{{\Delta\mathcal{E}}\; {l\left\lbrack H_{O} \right\rbrack}}\frac{1}{\lbrack G\rbrack}} + \frac{1}{{\Delta\mathcal{E}}\; {l\left\lbrack H_{O} \right\rbrack}}}} & (7)\end{matrix}$

Equation 7 suggests that a double reciprocal plot of ΔF and [G] shouldyield a straight line with x-intercept equal to −1/K_(d). FIG. 2 andFIG. 6 provide double reciprocal plots of the measured fluorescenceversus total concentration of compound [G_(O)]. Assuming that [G] can beapproximated as close to [G_(O)] (assumption 2), we can obtain estimatesfor the K_(d)'s of the compound-Aβ complexes from the x-intercept of thelinear fits of the data for each compound. The estimated K_(d)'s forsome compounds described herein are given in Table 3.

Example 8 ELISA Assay

Aggregated Aβ peptides were generated from synthetic Aβ(1-42) peptidesby dissolving 30 μg of peptide in 90 μL of nanopure water (pH 5-6) andincubating at 37° C. for ≧72 h without agitation. Each well of a 96-wellplate (well volume 0.4 mL; clear, flat bottom polypropylene) was coatedfor 3 h at 25° C. with 50 μL of 1.3 μM solution of Aβ peptides inphosphate-buffered saline (PBS, 10 mM NaH₂PO₄/Na₂HPO₄, 138 mM NaCl, 2.7mM KCl, pH 7.4). After removal of the excess sample, 50 μL solutions ofcompounds in PBS buffer (various concentrations were obtained bydiluting a stock solution with PBS buffer) were incubated in the wellsfor 12 h. Compounds that did not dissolve in PBS buffer were dissolvedin DMSO and diluted in PBS buffer to give a final solution of 5% DMSO inPBS buffer. The excess solutions were then removed and all wells wereblocked for 30 min by adding 300 μL of a 1% (w/v) solution of bovineserum albumin in PBS buffer (BSA/PBS). On occasion, an additionalblocking step was performed prior to incubation with solutions of smallmolecules. The blocking solution was discarded and the wells were washedonce with 300 μL of PBS buffer. Wells were incubated for 1 h with 50 μLof a 1.1 nM solution (in 1% BSA/PBS, dilution 1:6000) of anti-Aβ IgG(clone 6E10, monoclonal, mouse), followed by removal of the solution.The wells were washed twice with 300 μL of PBS buffer and incubated for60 min with 50 μL of the secondary IgG (anti-mouse IgG H+L, polyclonal,rabbit) conjugated with alkaline phosphatase (6.8 nM in 1% BSA/PBS,dilution 1:1000). The solution was discarded, and the wells were washedtwice with 300 μL PBS buffer. Bound secondary IgGs were detected by theaddition of 50 μL of a p-nitrophenyl phosphate solution (2.7 mM, in 100mM diethanol amine/0.5 mM magnesium chloride, pH 9.8). Absorbanceintensities were determined at 405 nm using a UV-vis spectroscopic platereader (Sprectramax 190, Molecular Devices, Sunnyvale, Calif.). Each runwas performed five times and averaged. Error bars represent standarddeviations. Graphs were plotted and fitted with the sigmoid curvefitting.

Example 9 Fluorescence Studies with Monomeric Aβ

Aβ (Biopeptide, Inc.) was initially solubilized in hexafluoroisopropanolat 1 mM concentration, vortexed, sonicated and vortexed. The vial wascovered in foil and was incubated for 21 hours at 25° C. on a shaker,with 3 times of vortexing throughout the incubation period. The solutionwas sonicated and vortexed again then diluted with cold nanopure water(2:1 H₂O:HFIP), fractionated in desired amounts into small glass vials,and immediately frozen in a CO₂/acetone bath. Each fraction was coveredwith parafilm that was punctured to allow solvent vapors to escape. Thefractions were lyophilized for 2 days to obtain monomeric Aβ (91%monomer by 12% Tris-bis PAGE gel analysis). 1.8 μL (8.42 μM) of thismonomeric Aβ(1-42) was added to 3 μL of 4 μM concentration of smallmolecules that was prepared by dissolving in PBS buffer pH 7.4 to attaina final concentration of 5 μM of Aβ(1-42) and 4 μM of the compound. Thesolution was transferred to 3 mL cuvettes and the fluorescence wasmeasured at 25° C.

Example 10 Evaluation of Rigid Rotors for Cytotoxic Activity AgainstSHSY-5Y Human Neuroblastoma Cells (MTT Assay)

SHSY-5Y human neuroblastoma cells, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cellproliferation kit, Eagle's Minimum Essential Medium (EMEM), Ham's F12nutrient mixture, and Fetal Bovine Serum (FBS) were all purchased fromATCC (Manassas, Va.). Briefly, SH-SY5Y cells (in 1:1 EMEM:Ham's F-12with 10% FBS) were seeded on 96-well plates at a density of 5×10⁴cells/well. Plates were incubated overnight (in a humidified atmosphereof 95% air, 5% CO₂, at 37° C.) to promote attachment of cells to thewells. Cells were then treated with various concentrations of compound8a, 8b, 8c, 8d, 11, or 14 and incubated for 24 hours (humidifiedatmosphere of 95% air, 5% CO₂, at 37° C.). MTT reagent (20 μL) was addedto the medium and incubated for additional 4 hours. After incubation,100 μL of detergent reagent was added and the plates were covered withaluminum foil and left at room temperature overnight. The amount ofsolubilized MTT formazan was measured by spectrophotometric absorbanceat 570 nm (Spectramax 190, Molecular Devices, Sunnyvale, Calif.). MTTassay was not performed on compound 19 due to its poor solubility inaqueous media. All data are presented as the mean±S.D, N=3 for eachconcentration. The Student's t-test was employed for all analyses. Ap-value of <0.05 was considered statistically significant compared tocontrol cells. As shown in FIG. 8, all compounds showed little or nocytotoxicity against human neuroblastoma cells at concentrations up to100 μM. These properties represent significant advantages for further invivo evaluation.

Example 11 Imaging Human Tissue with Amyloid Binding Compounds

FIGS. 11A-F depict fluorescence images of amyloid plaques in humantissue from AD cases. After the frozen tissue was sectioned and mountedto a glass slide, the tissue was exposed to a solution containing afluorescent probe for 30 min. The sample was washed with water toeliminate non-specific staining of the tissue, and imaged using aninverted epi-fluorescence microscope. The images reveal the location ofplaques that were stained with A) compound 27, B) compound 28, C)compound 29, D) compound 30, E) compound 31, or F) compound 33.

1.-16. (canceled)
 17. A method of detecting an amyloid peptide, themethod comprising: (i) contacting an amyloid peptide with a compoundhaving the structure of formula (I), or a pharmaceutical acceptable saltthereof, thereby forming a detectable amyloid complex; and (ii)detecting said detectable amyloid complex; wherein formula (I) has thestructure:

wherein EDG is an electron donor group; πCE is a pi-conjugation element;and WSG is a water soluble group; wherein said pi-conjugation elementhas the formula:-L¹-(A¹)_(q)-L²-(A²)_(r)-L³- or -L¹-(A¹)_(q)-L⁴-A³-L²-(A²)_(r)-L³-,wherein at least one of q or r is 1; at least one of A¹ or A² isR¹⁷-substituted or unsubstituted arylene or R¹⁷-substituted orunsubstituted heteroarylene; L¹, L², L³ and L⁴ are independently a bondor a linking group having the formula:

wherein x is an integer from 1 to 50; A³ is R¹⁷-substituted orunsubstituted arylene, or R¹⁷-substituted or unsubstitutedheteroarylene; R¹⁷ is halogen, —OR¹⁸, —NR¹⁹R²⁰, R²¹-substituted orunsubstituted alkyl, R²¹-substituted or unsubstituted heteroalkyl,R²¹-substituted or unsubstituted cycloalkyl, R²¹-substituted orunsubstituted heterocycloalkyl, R²¹-substituted or unsubstituted aryl,or R²¹-substituted or unsubstituted heteroaryl; R¹⁸, R¹⁹ and R²⁰ areindependently hydrogen, R²¹-substituted or unsubstituted alkyl,R²¹-substituted or unsubstituted heteroalkyl, R²¹-substituted orunsubstituted cycloalkyl, R²¹-substituted or unsubstitutedheterocycloalkyl, R²¹-substituted or unsubstituted aryl, orR²¹-substituted or unsubstituted heteroaryl; R²¹ is halogen, —OR²²,—NR²³R²⁴, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl; and R²², R²³ and R²⁴ are independentlyhydrogen or unsubstituted alkyl.
 18. The method of claim 17, wherein atleast one of A¹ or A² is R¹⁷-substituted or unsubstituted


19. The method of claim 17, wherein x is an integer from 1 to
 10. 20.The method of claim 17, wherein said water soluble group isR²⁵-substituted or unsubstituted alkyl, R²⁵-substituted or unsubstitutedheteroalkyl, R²⁵-substituted or unsubstituted cycloalkyl,R²⁵-substituted or unsubstituted heterocycloalkyl, R²⁵-substituted orunsubstituted aryl, R²⁵-substituted or unsubstituted heteroaryl; whereinR²⁵ is halogen, —OR²⁶, —NR²⁷R²⁸, R²⁹-substituted substituted orunsubstituted alkyl, R²⁹-substituted or unsubstituted heteroalkyl,R²⁹-substituted or unsubstituted cycloalkyl, R²⁹-substituted orunsubstituted heterocycloalkyl, R²⁹-substituted or unsubstituted aryl,or R²⁹-substituted or unsubstituted heteroaryl; R²⁶, R²⁷ and R²⁸ areindependently hydrogen, R²⁹-substituted or unsubstituted alkyl,R²⁹-substituted or unsubstituted heteroalkyl, R²⁹-substituted orunsubstituted cycloalkyl, R²⁹-substituted or unsubstitutedheterocycloalkyl, R²⁹-substituted or unsubstituted aryl, orR²⁹-substituted or unsubstituted heteroaryl, wherein R²⁷ and R²⁸ areoptionally joined together to form an R²⁹-substituted or unsubstitutedheterocycloalkyl, or R²⁹-substituted or unsubstituted heteroaryl; R²⁹ ishalogen, —OR³⁰, —NR³¹R³², unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, or unsubstituted heteroaryl; and R³⁰, R³¹ and R³²are independently hydrogen or unsubstituted alkyl.
 21. The method ofclaim 20, wherein said water soluble group is an ethylene glycol moietyhaving the formula:

wherein y is an integer from 1 to
 50. 22. The method of claim 21,wherein R²⁹ is unsubstituted alkyl.
 23. The method of claim 17, whereinsaid compound has the structure:


24. A method of detecting an amyloid peptide, the method comprising: (i)contacting an amyloid peptide with a compound having the structure offormula (IV), or a pharmaceutical acceptable salt thereof, therebyforming a detectable amyloid complex; and (ii) detecting said detectableamyloid complex; wherein formula (IV) has the structure:

wherein R⁴ and R⁵ are independently hydrogen, R¹²-substituted orunsubstituted alkyl, R¹²-substituted or unsubstituted heteroalkyl,R¹²-substituted or unsubstituted cycloalkyl, R¹²-substituted orunsubstituted heterocycloalkyl, R¹²-substituted or unsubstituted aryl orR¹²-substituted or unsubstituted heteroaryl, wherein R⁴ and R⁵ areoptionally joined together to form an R¹²-substituted or unsubstitutedheterocycloalkyl, or R¹²-substituted or unsubstituted heteroaryl; R¹² ishalogen, —OR¹³, —NR¹⁴R¹⁵, R¹⁶-substituted or unsubstituted alkyl,R¹⁶-substituted or unsubstituted heteroalkyl, R¹⁶-substituted orunsubstituted cycloalkyl, R¹⁶-substituted or unsubstitutedheterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, orR¹⁶-substituted or unsubstituted heteroaryl; R¹³, R¹⁴ and R¹⁵ areindependently hydrogen or unsubstituted alkyl; R¹⁶ is unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl; R¹⁷ is halogen, —OR¹⁸, —NR¹⁹R²⁰, R²¹-substituted orunsubstituted alkyl, R²¹-substituted or unsubstituted heteroalkyl,R²¹-substituted or unsubstituted cycloalkyl, R²¹-substituted orunsubstituted heterocycloalkyl, R²¹-substituted or unsubstituted aryl,or R²¹-substituted or unsubstituted heteroaryl; R¹⁸, R¹⁹ and R²⁰ areindependently hydrogen, R²¹-substituted or unsubstituted alkyl,R²¹-substituted or unsubstituted heteroalkyl, R²¹-substituted orunsubstituted cycloalkyl, R²¹-substituted or unsubstitutedheterocycloalkyl, R²¹-substituted or unsubstituted aryl, orR²¹-substituted or unsubstituted heteroaryl; R²¹ is halogen, —OR²²,—NR²³R²⁴, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, orunsubstituted heteroaryl; R²², R²³ and R²⁴ are independently hydrogen orunsubstituted alkyl; R²⁹ is halogen, —OR³⁰, —NR³¹R³², unsubstitutedalkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstitutedheteroaryl; R³⁰, R³¹ and R³² are independently hydrogen or unsubstitutedalkyl; y is an integer from 1 to 10; and z is an integer from 0 to 6.25. The method of claim 24, wherein said compound has the structure:


26. The method of claim 24, wherein R⁴ and R⁵ are optionally joinedtogether to form an R¹²-substituted or unsubstituted heterocycloalkyl.27. The method of claim 24, wherein said R¹²-substituted orunsubstituted heterocycloalkyl is a substituted or unsubstitutedpiperidinyl.
 28. The method of claim 24, wherein y is 2 and z is
 0. 29.The method of claim 17, wherein said amyloid peptide is Aβ peptide,prion, protein, α-synuclein, or superoxide dismutase.
 30. The method ofclaim 29, wherein said amyloid peptide forms part of an amyloid.